Anti-Stem Cell Factor (SCF) and Anti-Thymic Stromal Lymphocyte Poisoning Factor (TSLP) Antibodies and Bispecific Constructs

Abstract

Provided herein are novel anti-TSLP and anti-SCF antibodies, and their binding domains, and bispecific constructs comprising such antibodies and binding domains. Also provided herein are methods of treating disorders (such as disorders of the immune system) associated with an immune response (e.g., immune cell migration, activation, and / or proliferation) mediated by the interaction (e.g., binding) of TSLP and / or SCF with their receptors on immune cells (TSLPR and / or c-Kit, respectively) by administering to a patient in need thereof an antibody (or antigen-binding fragment thereof), bispecific construct, or composition described herein.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 488,388 (filed March 3, 2023), U.S. Provisional Patent Application No. 63 / 452,326 (filed March 15, 2023), and U.S. Provisional Patent Application No. 63 / 465,970 (filed May 12, 2023), the disclosures of which are incorporated by reference herein in their entireties. [Background technology]

[0002] I. Background of the Invention Inflammation plays a major role in many diseases, some of which are becoming more common and severe. Chronic inflammatory diseases contribute to more than half of deaths worldwide (Furman, D. et al., Nature Medicine, 2019, 25(12):1822-1832). Stem cell factor (SCF) and its receptor c-Kit are involved in perpetuating chronic inflammation.

[0003] SCF is expressed by various structural and inflammatory cells in the airways. Binding of SCF to c-Kit (also known as Kit ligand) leads to activation of multiple pathways, including the phosphatidylinositol-3 (PI3)-kinase pathway, the phospholipase C (PLC)-gamma pathway, the Src kinase pathway, the Janus kinase (JAK) / signal transducer and activator of transcription (STAT) pathway, and the mitogen-activated protein (MAP) kinase pathway (Reber, L. et al., Eur J Pharmacol, 2006, 533(1-3):327-40). SCF is expressed in two transmembrane forms (i.e., SCF α and SCF β) as a result of alternative splicing of exon 6. 220 and SCF 248 ) (Lennartsson and Roennstrand, Physiol Rev. 2012 Oct; 92(4):1619-49). SCF 248In SCF3, exon 6 is retained and encodes a proteolytic cleavage site that, when cleaved, results in soluble SCF3. 165 is born. SCF 220 lacks the cleavage site and forms a membrane-bound SCF dimer (mSCF).

[0004] c-Kit is a type III receptor tyrosine kinase that is encoded by c-kit gene.c-Kit comprises five extracellular immunoglobulin (Ig)-like domains, a single transmembrane domain, an inhibitory cytoplasmic juxtamembrane domain, and a split cytoplasmic kinase domain separated by a kinase insert segment (see, for example, Yarden et al., Nature, 1986, 323:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464).The human c-kit gene encoding c-Kit receptor has been cloned as described by Yarden et al., EMBO J., 1987, 6:3341-3351. c-Kit is also known as CD117 or stem cell factor receptor ("SCFR") because it is the receptor for stem cell factor (also known as Kit ligand). Binding of an SCF ligand to the first three extracellular Ig-like domains of c-Kit induces receptor dimerization, thereby activating intrinsic tyrosine kinase activity through phosphorylation of specific tyrosine residues in the juxtamembrane domain and kinase domain (see, e.g., Weiss and Schlessinger, Cell, 1998, 94:277-280; Clifford et al., J. Biol. Chem., 2003, 278:31461-31464). Members of the Stat, Src, ERK, and AKT signaling pathways have been shown to be downstream signaling factors of c-Kit signaling. The fourth (D4) and fifth (D5) extracellular Ig-like domains of c-Kit are thought to mediate receptor dimerization (see, e.g., International Patent Application Publication No. 2008 / 153926; Yuzawa et al., Cell, 2007, 130:323-334).

[0005] Expression of c-Kit has been detected in various cell types, such as mast cells, stem cells, brain cells, melanoblasts, ovarian cells, and cancer cells (e.g., leukemia cells) (see, e.g., Besmer, P. Curr. Opin. Cell Biol, 1991, 3:939-946; Lyman et al., Blood, 1998, 91: 1101-1134; Ashman, LK, Int. J. Biochem. Cell Biol, 1999, 31: 1037-1051; Kitamura et al., Mutat. Res., 2001, 477: 165-171; Mol et al., J. Biol. Chem., 2003, 278:31461-31464). Moreover, c-Kit plays an important role in hematopoiesis, melanogenesis, and gametogenesis (see Ueda et al., Blood, 2002, 99:3342-3349).

[0006] Mast cells are long-lived innate immune sentinel cells that reside in tissues throughout the body, particularly at the interface with the external environment (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00:1-11). Under normal homeostasis, mast cells can exert protective functions against helminth infections, venom, and may play a role in wound healing and initiation of adaptive responses. However, mast cells are better known for their role in initiating or contributing to numerous allergic, inflammatory, and autoimmune disorders.

[0007] Upon stimulation, mast cells release preformed mediators (proteases, histamine, serotonin, and cytokines) stored in granules, followed by a second wave of eicosanoids (leukotrienes and prostaglandin D2) and various proinflammatory cytokines and chemokines through de novo synthesis. These events lead to a rapid inflammatory response characterized by vasodilation, extravasation, smooth muscle contraction, pruritus, and recruitment of additional immune cell types, which can manifest in both acute and chronic conditions (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00:1-11).

[0008] Mast cell activation underlies the pathogenesis of allergic responses and is strongly associated with chronic, acute, and pruritic conditions, neuroinflammatory disorders, pain, fibrosis, and autoimmune diseases. Indeed, therapies that inhibit specific mast cell triggers, such as anti-IgE (omalizumab) or mediators (antihistamines), have been approved by health authorities and recommended by guidelines, yet many patients have limited benefit, suggesting that additional mast cell triggers or mediators are likely involved. Thus, therapies that lead to comprehensive mast cell suppression may offer broader efficacy in indications in which mast cells contribute to disease pathophysiology (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00:1-11).

[0009] The c-Kit (c-KIT / CD117) receptor tyrosine kinase and its sole ligand, stem cell factor (SCF), are master regulators of mast cell biology. c-Kit is highly expressed throughout the lifespan of mast cells and is also expressed in hematopoietic stem cells, melanocytes, interstitial cells of Cajal, germ cells, and a subset of taste receptor cells. Mast cells arise from multipotent hematopoietic stem cell precursors, enter the circulation as immature progenitors, and enter tissues, where they reach maturity. Phosphorylation of c-Kit by soluble or transmembrane SCF expressed on stromal cells (e.g., fibroblasts, keratinocytes, and endothelial cells) and on mast cells themselves regulates their differentiation, tissue migration, adhesion, maturation, and survival, and modulates their activation. Similarly, exogenous SCF is required for the differentiation, maturation, and maintenance of primary mast cells grown in vitro (Alvarado D, Maurer M, Gedrich R, et al. Allergy. 2022;00:1-11).

[0010] Thymic stromal lymphopoietin (TSLP) has also been shown to play a crucial role in initiating inflammation (Rui He and Raif S. Geha, Ann NY Acad Sci, 2010, 1183:13-24). TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine, and tonsils, as well as airway smooth muscle cells, lung fibroblasts, and interstitial cells (Reche et al., Journal of Immunology, 2001, 167: 336-343). These cells produce TSLP in response to proinflammatory stimuli, and TSLP initiates allergic inflammatory responses through its activity on several innate immune cells, including dendritic cells, monocytes, and mast cells (Soumelis et al., Nature Immunology, 2002, 3:673-680; Reche et al., Journal of Immunology, 2001, 167:336-343; (Allakhverdi et al. (2007) The Journal of Experimental Medicine 204:253-258). TSLP has also been shown to play a role in the pathogenesis of diseases, including fibrosis. For example, TSLP has been shown to be upregulated in fibrotic conditions in both the skin and lung (Shin et al., Journal of Investigative Dermatology, 2016, 136(2):360-362).

[0011] Despite therapeutic advances, there is a need in the art for new and improved agents for treating conditions or diseases associated with inflammation. Summary of the Invention

[0012] II. Summary of the Invention Provided herein are anti-TSLP and anti-SCF antibodies (e.g., fully human antibodies, humanized antibodies, and chimeric antibodies), as well as binding domains (i.e., antigen-binding fragments) thereof. Also provided are bispecific and multispecific constructs comprising anti-TSLP and / or anti-SCF antibodies (or antigen-binding fragments thereof) linked to at least one additional binding agent (e.g., a ligand or antibody or antigen-binding fragment thereof). In one embodiment, the bispecific and multispecific constructs comprise the anti-TSLP and / or anti-SCF antibodies (or antigen-binding fragments thereof) described herein linked to at least one additional binding agent. Compositions comprising the antibodies, bispecific and multispecific constructs are also provided.

[0013] Also provided are methods for treating inflammatory diseases or conditions associated with the expression and / or activity of TSLP and / or SCF, e.g., diseases or conditions associated with the migration, activation, and / or proliferation of immune cells via the interaction of TSLP with its receptor on immune cells (TSLPR) and / or SCF with its receptor on immune cells (c-Kit). In some embodiments, the present disclosure provides methods for inhibiting or blocking the binding of TSLP to TSLPR and / or blocking the binding of SCF to c-Kit, for inhibiting or preventing immune cell activation, and for reducing or preventing the accumulation of immune cells in organs or tissues, thereby treating or preventing various diseases and disorders involving inflammation. Also provided herein are methods of reducing inflammation in a subject in need thereof, as well as methods of treating inflammatory diseases or disorders (e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, pulmonary diseases, metabolic diseases (such as type 2 diabetes), neurodegenerative diseases (such as Parkinson's disease), certain types of cancer (such as colon cancer), and psychiatric illnesses (such as depression)) by administering to a patient in need thereof a bispecific or multispecific construct, antibody or antigen-binding fragment thereof, or composition described herein.

[0014] In one embodiment, the anti-TSLP antibody or binding domain thereof comprises the heavy and / or light chain CDRs or variable regions of any one of antibodies 1D10-A, 1D10-B, 1D10-C, 1D10-D, 1D10-E, 1D10-F, 1D10-G, 1D10-H, or 1D10-I (as shown in Table 6 in Example 2). In another embodiment, the anti-TSLP antibody or binding domain thereof comprises the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1, 5, 9, or 13, and / or the CDR1, CDR2, and CDR3 domains of a light chain variable region having the amino acid sequence set forth in SEQ ID NO:17, 21, 25, or 29. In another embodiment, the anti-TSLP antibody or binding domain thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 5A (in Example 2) or conservative sequence modifications thereof, respectively, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 5B (in Example 2) or conservative sequence modifications thereof, respectively. In another embodiment, the anti-TSLP antibody or binding domain thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:2, 3, and 4, (b) SEQ ID NOs:6, 7, and 8, (c) SEQ ID NOs:10, 11, and 12, and (d) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:18, 19, and 20, (b) SEQ ID NOs:22, 23, and 24, (c) SEQ ID NOs:26, 27, and 28, and (d) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof, as set forth in Table 1A below.In another embodiment, the anti-TSLP antibody or binding domain comprises the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:6, 7, and 8 (i.e., the heavy chain CDR1, CDR2, and CDR3 sequences of 1D10-H2), and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:18, 19, and 20 (i.e., the light chain CDR1, CDR2, and CDR3 sequences of 1D10-L1), respectively.

[0015] Table 1A. Anti-TSLP heavy and light chain CDR pairing TIFF2026508389000001.tif129160

[0016] In another embodiment, the anti-TSLP antibody or binding domain thereof comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:1, 5, 9, or 13, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the antibody or binding domain thereof comprises a light chain variable region having an amino acid sequence set forth in SEQ ID NO:17, 21, 25, or 29, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0017] In another embodiment, the anti-TSLP antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 1B below. In one embodiment, the anti-TSLP antibody or binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:5 (i.e., the heavy chain variable region of 1D10-H2) and a light chain variable region having the amino acid sequence of SEQ ID NO:17 (i.e., the light chain variable region of 1D10-L1).

[0018] Table 1B. Anti-TSLP heavy and light chain VH / VL pairings TIFF2026508389000002.tif63159

[0019] In certain embodiments, the anti-TSLP antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 6 of Example 2.

[0020] In one embodiment, the anti-SCF antibody or binding domain thereof comprises the heavy and / or light chain CDRs or variable regions of any one of anti-SCF antibodies mAb12-A, mAb12-B, mAb12-C, mAb12-D, mAb12-E, mAb12-F, mAb12-G, mAb12-H, or mAb12-I (as shown in Table 9 in Example 9). In another embodiment, the anti-SCF antibody or binding domain thereof comprises the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:33, 37, 41, or 45, and / or the CDR1, CDR2, and CDR3 domains of a light chain variable region having the amino acid sequence set forth in SEQ ID NO:49, 53, 57, or 61. In another embodiment, the anti-SCF antibody or binding domain thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 8A (in Example 9) or conservative sequence modifications thereof, respectively, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 8B or conservative sequence modifications thereof, respectively. In another embodiment, the anti-SCF antibody or binding domain thereof comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:34, 35, and 36, (b) SEQ ID NOs:38, 39, and 40, (c) SEQ ID NOs:42, 43, and 44, and (d) SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:50, 51, and 52, (b) SEQ ID NOs:54, 55, and 56, (c) SEQ ID NOs:58, 59, and 60, and (d) SEQ ID NOs:62, 63, and 64, respectively, or conservative sequence modifications thereof, as set forth in Table 2A below.In one embodiment, the anti-SCF antibody or binding domain comprises the heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:46, 47, and 48 (i.e., the heavy chain CDR1, CDR2, and CDR3 sequences of mAb12-H4), and the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:58, 59, and 60 (i.e., the light chain CDR1, CDR2, and CDR3 sequences of mAb12-L3).

[0021] Table 2A. Anti-SCF heavy and light chain CDR pairings TIFF2026508389000003.tif124166

[0022] In another embodiment, the antibody or binding domain thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:33, 37, 41, or 45, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the antibody or binding domain thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:49, 53, 57, or 61, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0023] In another embodiment, the anti-SCF antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 2B below. In one embodiment, the anti-SCF antibody or binding domain comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO:45 (i.e., the heavy chain variable region of mAb12-H4) and a light chain variable region having the amino acid sequence of SEQ ID NO:57 (i.e., the light chain variable region of mAb12-L3).

[0024] Table 2B: Anti-SCF heavy and light chain VH / VL pairings TIFF2026508389000004.tif67170

[0025] In certain embodiments, the anti-SCF antibody or binding domain thereof comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 9 of Example 9.

[0026] In one embodiment, the CDR1, CDR2, and / or CDR3 regions of an anti-TSLP antibody or binding domain described herein comprise the amino acid sequence of antibody 1D10 (i.e., heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:2, 3, and 4; (b) SEQ ID NOs:6, 7, and 8; (c) SEQ ID NOs:10, 11, and 12; and (d) SEQ ID NOs:14, 15, and 16, respectively; and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:18, 19, and 20; (b) SEQ ID NOs:22, 23, and 24; (c) SEQ ID NOs:26, 27, and 28; and (d) SEQ ID NOs:30, 31, and 32, respectively).

[0027] In one embodiment, the CDR1, CDR2, and / or CDR3 regions of the anti-SCF antibody or binding domain described herein comprise the amino acid sequence of antibody mAb12 disclosed herein (i.e., heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:34, 35, and 36; (b) SEQ ID NOs:38, 39, and 40; (c) SEQ ID NOs:42, 43, and 44; (d) SEQ ID NOs:46, 47, and 48, respectively; and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:50, 51, and 52; (b) SEQ ID NOs:54, 55, and 56; (c) SEQ ID NOs:58, 59, and 60; (d) SEQ ID NOs:62, 63, and 64, respectively).

[0028] In another embodiment, the antibody comprises derivatives of the CDR sequences of 1D10 and mAb12, while still retaining the ability to effectively bind to either TSLP or SCF. Such derivatives include CDRs that contain one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid additions, deletions, or substitutions, e.g., conservative sequence substitutions.

[0029] In another embodiment, the anti-TSLP or anti-SCF antibody or binding domain comprises one or more CDRs that are 90%, 95%, 98%, or 99.5% identical to one or more CDRs of, for example, antibody 1D10 and antibody mAb12, respectively. CDRs with identities intermediate to the above-listed values, e.g., 90-95%, 95-98%, or 98-100% identical to one or more of the above sequences. The antibody or binding domain sequence can also comprise a consensus sequence.

[0030] Also encompassed are sequences substantially identical to the anti-TSLP antibody and / or anti-SCF antibody sequences or anti-TSLP binding domain and / or anti-SCF binding domain sequences described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the foregoing sequences). In one embodiment, the anti-TSLP antibody or binding domain comprises a heavy chain variable region comprising SEQ ID NO: 1, 5, 9, 13, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the anti-TSLP binding domain comprises a light chain variable region comprising SEQ ID NO: 17, 21, 25, 29, 18, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0031] In another embodiment, the anti-SCF antibody or binding domain comprises a heavy chain variable region comprising SEQ ID NO:33, 37, 41, 45, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto). In another embodiment, the anti-SCF antibody or binding domain comprises a light chain variable region comprising SEQ ID NO:49, 53, 57, 61, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0032] Also encompassed are anti-TSLP and / or anti-SCF antibodies or binding domains thereof that compete for binding with any of the antibodies or binding domains thereof described herein or that bind to the same epitope as any of the antibodies or binding domains described herein. For example, in one embodiment, an anti-TSLP antibody or binding domain thereof competes with antibody 1D10 described herein for binding to TSLP. In another embodiment, an anti-TSLP antibody or binding domain thereof binds to the same epitope on TSLP as antibody 1D10 described herein. In another embodiment, an anti-SCF antibody or binding domain thereof competes with antibody mAb12 described herein for binding to SCF. In another embodiment, an anti-SCF antibody or binding domain thereof binds to the same epitope on SCF as antibody mAb12 described herein. In another embodiment, an anti-SCF antibody or binding domain thereof binds to membrane-bound human SCF (hSCF 222 soluble human SCF (hSCF) 165 In another embodiment, the anti-SCF antibody or binding domain binds to an epitope comprising residue K100 of human SCF.

[0033] In one embodiment, a bispecific or multispecific construct provided herein comprises an anti-TSLP antibody (or binding domain) or an anti-SCF antibody (or binding domain) linked to a second binding agent (e.g., a ligand, a second antibody, or an antigen-binding domain thereof). In another embodiment, the construct comprises an anti-TSLP antibody (or binding domain) or an anti-SCF antibody (or antigen-binding domain) comprising the heavy and light chain CDR sequences and / or full-length variable region sequences set forth in Tables 1A, 1B, and 2A, 2B, respectively. In yet another embodiment, the second antibody (or binding domain) binds to a member of the TNF superfamily (e.g., TNFα), a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4), an interleukin (e.g., IL-23, IL-23A, IL-17A, IL-5, IL-11, IL-12, or IL-13), an immunoglobulin (e.g., IgE), or an integrin (e.g., integrin α4β7), or OX40L, or VEGF. The term "bispecific construct," as used herein, also refers to a "multispecific construct" that includes a third, fourth, or more antibodies (or fragments thereof).

[0034] In one such embodiment, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a member of the TNF superfamily (e.g., TNFα; e.g., the variable domain of adalimumab, golimumab, or certolizumab).

[0035] In a further embodiment, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4).

[0036] In a further aspect, a construct is provided comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-12 and / or IL-23 (e.g., the variable domain of guselkumab, tildrakizumab, or ustekinumab).

[0037] In a further aspect, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-23A (e.g., the variable domain of risankizumab).

[0038] In a further aspect, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-17A (e.g., the variable domain of secukinumab).

[0039] In a further aspect, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-13 (e.g., the variable domain of lebrikizumab or tralokinumab).

[0040] In a further aspect, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IgE (e.g., the variable domain of omalizumab or ligelizumab).

[0041] In a further embodiment, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-5.

[0042] In a further embodiment, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-11.

[0043] In a further aspect, a construct is provided comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to CD40L (e.g., the variable domain of oxelumab; see U.S. Patent No. 7,501,496).

[0044] In a further aspect, a construct is provided comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to VEGF (e.g., the variable domain of bevacizumab; see U.S. Patent No. 7,060,269).

[0045] In a further aspect, a construct is provided that comprises an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to an integrin (e.g., integrin α4β7; e.g., the variable domain of vedolizumab).

[0046] In a further embodiment, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a member of the TNF superfamily (e.g., TNFα; e.g., the variable domain of adalimumab, golimumab, or certolizumab).

[0047] In a further embodiment, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-12 and / or IL-23 (e.g., the variable domain of guselkumab, tildrakizumab, or ustekinumab).

[0048] In a further embodiment, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-23A (e.g., the variable domain of risankizumab).

[0049] In a further aspect, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-17A (e.g., the variable domain of secukinumab).

[0050] In a further aspect, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-13 (e.g., the variable domain of lebrikizumab or tralokinumab).

[0051] In a further embodiment, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds IgE (e.g., the variable domain of omalizumab or ligelizumab).

[0052] In a further embodiment, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to an integrin (e.g., integrin α4β7; e.g., the variable domain of vedolizumab).

[0053] In a further aspect, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to CD40L (e.g., the variable domain of oxelumab; see U.S. Patent No. 7,501,496).

[0054] In a further aspect, a construct is provided that includes an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to VEGF (e.g., the variable domain of bevacizumab; see U.S. Patent No. 7,060,269).

[0055] In another embodiment, the anti-TSLP antibody (or antigen-binding domain) of the bispecific or multispecific construct comprises the amino acid sequences of the heavy and / or light chain CDRs and / or full-length variable regions set forth in Tables 1A and 1B, respectively. In another embodiment, the anti-SCF antibody (or antigen-binding domain) of the bispecific or multispecific construct comprises the amino acid sequences of the heavy and / or light chain CDRs and / or full-length variable regions set forth in Tables 2A and 2B.

[0056] In another embodiment, the bispecific or multispecific construct comprises an anti-TSLP antibody (or binding domain) and an anti-SCF antibody (or binding domain) linked to each other. Thus, further provided is a construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds TSLP.

[0057] In certain embodiments, the anti-SCF and / or anti-TSLP antibodies or anti-SCF and / or anti-TSLP binding domains are antibodies or binding domains having the CDR and / or variable domain sequences described herein.

[0058] In one embodiment, the bispecific construct comprises: (a) an anti-TSLP binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 5A or conservative sequence modifications thereof, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 5B or conservative sequence modifications thereof; and (b) an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 8A or conservative sequence modifications thereof, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 8B or conservative sequence modifications thereof.

[0059] In another embodiment, the bispecific construct comprises: (a) an anti-TSLP binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:2, 3, and 4, (ii) SEQ ID NOs:6, 7, and 8, (iii) SEQ ID NOs:10, 11, and 12, and (iv) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:18, 19, and 20, (ii) SEQ ID NOs:22, 23, and 24, (iii) SEQ ID NOs:26, 27, and 28, and (iv) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof; and (b) an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:34, 35, and 36, (ii) SEQ ID NOs:38, 39, and 40, (iii) SEQ ID NOs:42, 43, and 44, and (iv) SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:50, 51, and 52, (ii) SEQ ID NOs:54, 55, and 56, (iii) SEQ ID NOs:58, 59, and 60, and (iv) SEQ ID NOs:62, 63, and 64, respectively, or conservative sequence modifications thereof.

[0060] In one embodiment, the bispecific construct comprises: (a) an anti-TSLP binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, or conservative sequence modifications thereof, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, or conservative sequence modifications thereof; and (b) an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs:58, 59, and 60, respectively, or conservative sequence modifications thereof.

[0061] In another embodiment, the bispecific construct comprises a combination of anti-TSLP heavy chain variable region sequences and anti-TSLP light chain variable region sequences having an amino acid sequence set forth in Table 1B or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto), and anti-SCF heavy chain variable region sequences and anti-SCF light chain variable region sequences having an amino acid sequence set forth in Table 2B or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0062] In another embodiment, the bispecific construct comprises an anti-TSLP heavy chain variable region sequence and an anti-TSLP light chain variable region sequence having an amino acid sequence set forth in Table 4 or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto), and an anti-SCF heavy chain variable region sequence and / or an anti-SCF light chain variable region sequence having an amino acid sequence set forth in Table 5 or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0063] In another embodiment, the bispecific construct comprises: (a) an anti-TSLP binding domain comprising a combination of heavy chain and light chain variable region sequences having an amino acid sequence set forth in Table 1B or a sequence at least 90% identical thereto; and (b) an anti-SCF binding domain comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:34, 35, and 36, (ii) SEQ ID NOs:38, 39, and 40, (iii) SEQ ID NOs:42, 43, and 44, and (iv) SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:50, 51, and 52, (ii) SEQ ID NOs:54, 55, and 56, (iii) SEQ ID NOs:58, 59, and 60, and (iv) SEQ ID NOs:62, 63, and 64, respectively, or conservative sequence modifications thereof.

[0064] In another embodiment, the bispecific construct comprises: (a) an anti-SCF binding domain comprising a combination of heavy chain and light chain variable region sequences having an amino acid sequence set forth in Table 2B or a sequence at least 90% identical thereto; and (b) An anti-TSLP binding domain comprising heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:2, 3, and 4, (ii) SEQ ID NOs:6, 7, and 8, (iii) SEQ ID NOs:10, 11, and 12, and (iv) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:18, 19, and 20, (ii) SEQ ID NOs:22, 23, and 24, (iii) SEQ ID NOs:26, 27, and 28, and (iv) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof.

[0065] In another embodiment, the bispecific construct comprises an anti-TSLP-binding domain and an anti-SCF-binding domain comprising the amino acid sequences set forth in Table 10 of Example 19, e.g., construct 5.7 (1D10(VH1-L1)-mAb12(VH4-L3)), also referred to herein as CDX-622. In one embodiment, the anti-TSLP-binding domain and the anti-SCF-binding domain are genetically fused. The bispecific construct can be, for example, a fusion protein, which can be generated by genetic engineering using standard recombinant DNA techniques to operably link nucleic acids encoding the anti-TSLP7-binding domain and the anti-SCF-binding domain. In another embodiment, the anti-TSLP-binding domain and the anti-SCF-binding domain are chemically conjugated.

[0066] For example, the bispecific construct can be a conjugate created by chemical conjugation of the anti-TSLP-binding domain and the anti-SCF-binding domain. In one embodiment, the anti-TSLP-binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-SCF-binding domain is linked to the C-terminus of the heavy chain of the anti-TSLP-binding domain. In another embodiment, the anti-SCF-binding domain is an scFv.

[0067] In another embodiment, the anti-TSLP binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-SCF binding domain is linked to the C-terminus of the heavy chain of the anti-TSLP binding domain. In another embodiment, the anti-SCF binding domain is an scFv.

[0068] In another embodiment, the anti-SCF binding domain further comprises a human IgG1 constant domain. In another embodiment, the anti-TSLP binding domain is linked to the C-terminus of the heavy chain of the anti-SCF binding domain. In another embodiment, the anti-TSLP binding domain is an scFv.

[0069] In certain embodiments, the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) an anti-TSLP antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:2, 3, and 4, (ii) SEQ ID NOs:6, 7, and 8, (iii) SEQ ID NOs:10, 11, and 12, and (iv) SEQ ID NOs:14, 15, and 16, respectively, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:18, 19, and 20, (ii) SEQ ID NOs:22, 23, and 24, (iii) SEQ ID NOs:26, 27, and 28, and (iv) SEQ ID NOs:30, 31, and 32, respectively; (b) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:34, 35, and 36, (ii) SEQ ID NOs:38, 39, and 40, (iii) SEQ ID NOs:42, 43, and 44, or (iv) SEQ ID NOs:46, 47, and 48, respectively, and / or light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:50, 51, and 52, (ii) SEQ ID NOs:54, 55, and 56, (iii) SEQ ID NOs:58, 59, and 60, or (iv) SEQ ID NOs:62, 63, and 64, respectively; and and, (c) Human IgG1 constant domain.

[0070] In one embodiment, the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) an anti-TSLP antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs:6, 7, and 8, respectively, and light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs:18, 19, and 20, respectively; (b) an anti-SCF scFv comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively; and and, (c) Human IgG1 constant domain.

[0071] In another specific embodiment, the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) an anti-SCF antibody comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:34, 35, and 36, (ii) SEQ ID NOs:38, 39, and 40, (iii) SEQ ID NOs:42, 43, and 44, or (iv) SEQ ID NOs:46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:50, 51, and 52, (ii) SEQ ID NOs:54, 55, and 56, (iii) SEQ ID NOs:58, 59, and 60, or (iv) SEQ ID NOs:62, 63, and 64, respectively, and a human IgG1 constant domain; and (b) The anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:2, 3, and 4, (ii) SEQ ID NOs:6, 7, and 8, (iii) SEQ ID NOs:10, 11, and 12, and (iv) SEQ ID NOs:14, 15, and 16, respectively, and light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:18, 19, and 20, (ii) SEQ ID NOs:22, 23, and 24, (iii) SEQ ID NOs:26, 27, and 28, and (iv) SEQ ID NOs:30, 31, and 32, respectively.

[0072] In one embodiment, the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (c) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively, and a human IgG1 constant domain; and (d) The anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs:6, 7, and 8, respectively, and light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs:18, 19, and 20, respectively.

[0073] In certain embodiments, the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) an anti-TSLP antibody comprising a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 1B, and a human IgG1 constant domain; and (b) Anti-SCF scFvs comprise a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 2B.

[0074] In another specific embodiment, the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) an anti-SCF antibody comprising a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 2B, and a human IgG1 constant domain; and (b) The anti-TSLP scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 1B.

[0075] In certain embodiments, the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) an anti-TSLP antibody comprising a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 4, and a human IgG1 constant domain; and (b) Anti-SCF scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 5.

[0076] In another specific embodiment, the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) an anti-SCF antibody comprising a combination of heavy chain and light chain variable region sequences having the amino acid sequences set forth in Table 5, and a human IgG1 constant domain; and (b) Anti-TSLP scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 4.

[0077] In certain embodiments, the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) an anti-TSLP antibody comprising a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 6, and a human IgG1 constant domain; and (b) Anti-SCF scFv comprises a combination of heavy and light chain variable region sequences having the amino acid sequences set forth in Table 6.

[0078] An exemplary bispecific is Construct 5.2 (1D10 H2-mAb 12 VH1VL4(ds)), having the amino acid sequence set forth in SEQ ID NO:65 or encoded by the nucleotide sequence set forth in SEQ ID NO:66.

[0079] Another exemplary bispecific is construct 5.7 (1D10 H2-mAb12 VH4VL3(ds)), also referred to herein as CDX-622, having the amino acid sequence set forth in SEQ ID NO:67 or encoded by the nucleotide sequence set forth in SEQ ID NO:68.

[0080] Also provided herein are compositions comprising any of the antibodies, antigen-binding fragments, or bispecific constructs described herein and a pharmaceutically acceptable carrier. Further provided are kits or vials containing any of the antibodies, antigen-binding fragments, or bispecific constructs described herein, and instructions for use.

[0081] In a further aspect, isolated nucleic acid molecules encoding the antibodies, antigen-binding fragments, or bispecific constructs described herein, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors, are also provided.

[0082] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region comprises an amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the foregoing sequences).

[0083] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and light chain variable regions of the antibody, wherein the heavy and light chain variable regions comprise an amino acid sequence set forth in Table 1B or Table 2B, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0084] Also provided herein are in vitro and in vivo methods for blocking or inhibiting the binding of TSLP and / or SCF to their receptors (i.e., TLSPR and c-Kit, respectively), comprising contacting a cell expressing TSLPR or c-Kit with an antibody, antigen-binding fragment, or bispecific construct (or composition) described herein in an amount effective to block the binding of TSLP and / or SCF to its receptor.

[0085] In another aspect, methods are provided for inhibiting immune cell activation in a subject and reducing or preventing immune cell accumulation in an organ or tissue in a subject, comprising administering to the subject any of the antibodies, antigen-binding fragments, or bispecific constructs (or compositions) described herein in an amount effective to inhibit immune cell activation in the subject or reduce immune cell accumulation in an organ or tissue in the subject. In one embodiment, such methods result in inhibition or blockage of (a) TSLP-induced activation and / or proliferation of mast cells, DCs, and / or NKT cells, (b) TSLP-induced osteoprotegerin (OPG) secretion, (c) TSLP-induced secretion of Th2 cytokines (such as TARC, CCL22, IL-4, IL-13, or IL-5), and / or (d) SCF-induced secretion of mast cells, eosinophils, type 2 innate lymphoid cells (ILC2) cells, and / or type 3 innate lymphoid cells (ILC3) cells.

[0086] In another aspect, methods for reducing or inhibiting inflammation in a subject in need thereof by administering to a patient in need thereof an antibody, antigen-binding fragment, or bispecific construct (or composition) described herein in an amount effective to inhibit or reduce inflammation in the subject, as well as methods for treating an inflammatory disease or disorder (e.g., autoimmune disease, cardiovascular disease, gastrointestinal disease, pulmonary disease, metabolic disease (such as type 2 diabetes), vasculitis (Takayasu's arteritis, polymyalgia rheumatica, and the like). Provided herein are methods for treating diseases such as: large vasculitis, such as cerebrovascular disease and temporal arteritis; medium-sized vasculitis, such as Buerger's disease, Kawasaki disease, cutaneous vasculitis, and polyarteritis nodosa; and small vasculitis, such as Behcet's syndrome, Churg-Strauss syndrome, cutaneous vasculitis, Henoch-Schönlein purpura, granulomatosis with polyangiitis, microscopic polyangiitis, and cryoglobulinemia; neurodegenerative diseases (such as Parkinson's disease); certain types of cancer (such as colon cancer); and psychiatric disorders (such as depression).

[0087] In another aspect, a method for treating a condition or disease in a subject is provided, comprising administering to the subject any antibody, antigen-binding fragment, or bispecific construct (or composition) described herein in an amount effective to treat the condition or disease. The subject may be, for example, a subject suffering from a condition or disease in which reduced inflammation is desired. In one aspect, the condition or disease is associated with immune cell migration, activation, and / or proliferation via interaction (e.g., binding) of TSLP and / or SCF with receptors on immune cells (TSLPR and / or c-Kit, respectively), such as immune system disorders, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease (COPD), and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhinoconjunctivitis. Other conditions and disorders include fibrotic diseases and illnesses associated with tissue remodeling, such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, smoking-induced lung injury, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, liver fibrosis, fibromyalgia, gingival fibrosis, or radiation-induced fibrosis.

[0088] In another aspect, a method for treating a condition or disease in a subject is provided, wherein the method comprises administering to the subject a combination of any anti-TSLP antibody, or antigen-binding fragment thereof, described herein and any anti-SCF antibody, or antigen-binding fragment thereof, described herein. (i) an anti-TSLP antibody or antigen-binding fragment thereof, comprising heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:2, 3, and 4, (b) SEQ ID NOs:6, 7, and 8, (c) SEQ ID NOs:10, 11, and 12, and (d) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:18, 19, and 20, (b) SEQ ID NOs:22, 23, and 24, (c) SEQ ID NOs:26, 27, and 28, and (d) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof; and (ii) An anti-SCF antibody or antigen-binding fragment thereof, comprising heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:34, 35, and 36, (b) SEQ ID NOs:38, 39, and 40, (c) SEQ ID NOs:42, 43, and 44, and (d) SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:50, 51, and 52, (b) SEQ ID NOs:54, 55, and 56, (c) SEQ ID NOs:58, 59, and 60, and (d) SEQ ID NOs:62, 63, and 64, respectively, or conservative sequence modifications thereof.

[0089] In another aspect, (i) the anti-TSLP antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region amino acid sequence set forth in Table 1B, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto); and (ii) The anti-SCF antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region amino acid sequence set forth in Table 2B, or a sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0090] In one embodiment, the anti-TSLP antibody or antigen-binding fragment thereof and the anti-SCF antibody or antigen-binding fragment thereof are administered separately. In one embodiment, the anti-TSLP antibody or antigen-binding fragment thereof and the anti-SCF antibody or antigen-binding fragment thereof are administered sequentially. For example, the anti-TSLP antibody or antigen-binding fragment thereof can be administered first, followed (e.g., immediately thereafter) by the anti-SCF antibody or antigen-binding fragment thereof, or vice versa. In another embodiment, the anti-TSLP antibody or antigen-binding fragment thereof and the anti-SCF antibody or antigen-binding fragment thereof are administered together. In another embodiment, the anti-TSLP antibody or antigen-binding fragment thereof and the anti-SCF antibody or antigen-binding fragment thereof are administered simultaneously. In another embodiment, the anti-TSLP antibody or antigen-binding fragment thereof and the anti-SCF antibody or antigen-binding fragment thereof are administered simultaneously in a single formulation. Alternatively, the anti-TSLP antibody or antigen-binding fragment thereof and the anti-SCF antibody or antigen-binding fragment thereof are formulated for separate administration and administered concurrently or sequentially. Such concurrent or sequential administration preferably results in the simultaneous presence of both antibodies in the treated patient.

[0091] In one embodiment, the antibody, antigen-binding fragment, or bispecific construct (or composition described herein) is administered in combination with one or more additional therapeutic agents or procedures. [Brief explanation of the drawings]

[0092] III. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] 1 is a graph showing binding of anti-TSLP antibodies to human TSLP as a function of antibody concentration. [Figure 2]1 is a graph showing binding of anti-TSLP antibodies to human TSLP as a function of antibody concentration. [Figure 3] 1 is a graph showing binding of anti-TSLP antibodies to human TSLP as a function of antibody concentration. [Figure 4] 1 is a graph showing that anti-TSLP antibodies block the binding of human TSLP to the human TSLP receptor (TSLP-R). [Figure 5] 1 is a graph showing that anti-TSLP antibodies block the binding of human TSLP to the human TSLP receptor (TSLP-R). [Figure 6] 1 is a graph showing that anti-TSLP antibodies block the binding of human TSLP to the human TSLP receptor (TSLP-R). [Figure 7] 1 is a graph showing that anti-TSLP antibodies inhibit the proliferation of BaF3 cells. [Figure 8] 1 is a graph showing that anti-TSLP antibodies inhibit TARC induction in human dendritic cells. [Figure 9] Figures 9A and 9B are tables showing the affinity and kinetic parameters (background subtracted) of anti-TSLP antibodies for human (Figure 9A) and cynomolgus monkey (Figure 9B) TSLP. [Figure 10] 1 is a graph showing binding of anti-SCF antibodies to human SCF as a function of antibody concentration. [Figure 11] 1 is a graph showing binding of anti-SCF antibodies to human SCF as a function of antibody concentration. [Figure 12] 1 is a graph showing that anti-SCF antibodies block the binding of human SCF to human c-Kit. [Figure 13] 1 is a graph showing that anti-SCF antibodies block the binding of human SCF to human c-Kit. [Figure 14] 14A and 14B are tables showing the affinity and kinetic parameters (background subtracted) of anti-SCF antibodies for human (FIG. 14A) and cynomolgus monkey (FIG. 14B) SCF. [Figure 15]1 is a graph showing that anti-SCF antibodies inhibit phosphorylation of c-Kit in human CHO-KIT cells. [Figure 16] 1 is a graph showing that anti-SCF antibodies inhibit degranulation of human mast cells. [Figure 17] 1 is a graph showing that anti-SCF antibodies inhibit the proliferation of M-07e cells. [Figure 18A] Figures 18A and 18B are graphs showing how binding of anti-SCF to human SCF is affected by mutating amino acid residues in human SCF. [Figure 18B] See legend to Figure 18A. [Figure 18C] 1 is a three-dimensional representation showing the binding of SCF to KIT-D1, including residue K100. [Figure 19] 19A and 19B are graphs showing binding of anti-SCF antibodies to S1 / S14 mouse cells. [Figure 20] 1 is a graph showing that anti-SCF antibodies inhibit phosphorylation of c-Kit in human M-07e cells. [Figure 21] Figures 21A and 21B are schematic diagrams showing the structures of a bispecific construct with an anti-SCF antibody linked to an anti-TSLP scFv (A) and a bispecific construct with an anti-TSLP antibody linked to an anti-SCF scFv (B). [Figure 22] 1 is a graph showing binding of bispecific antibody constructs to human TSLP. [Figure 23] FIG. 1 is a graph showing binding of bispecific antibody constructs to human SCF. [Figure 24] 1 is a graph showing binding of bispecific antibody constructs to human TSLP and human SCF. [Figure 25] Figures 25A and 25B are graphs showing that bispecific antibody constructs block the binding of human TSLP to the human TSLP receptor (TSLP-R). [Figure 26]1 is a graph showing that bispecific antibody constructs block the binding of human SCF to c-Kit. [Figure 27] Figures 27A and 27B are graphs showing that bispecific antibody constructs inhibit the proliferation of BaF3 cells. [Figure 28] Figures 28A and 28B are graphs showing that bispecific antibody constructs inhibit TARC induction in human dendritic cells. [Figure 29] Figures 29A and 29B are tables showing the affinity and kinetic parameters (background subtracted) of bispecific antibody constructs against human TSLP and human SCF, and cynomolgus TSLP and cynomolgus SCF. [Figure 30] FIG. 1 is a graph showing that bispecific antibody constructs inhibit phosphorylation of c-Kit in human CHO-KIT cells. [Figure 31] FIG. 1 is a graph showing that bispecific antibody constructs inhibit human mast cell degranulation. [Figure 32] FIG. 1 is a graph showing that bispecific antibody constructs inhibit the proliferation of M-07e cells. [Figure 33] 1 is a graph showing that the bispecific antibody CDX-622 more potently blocks phosphorylation of KIT in M-07e cells stimulated with soluble SCF than KIT stimulated in SCF220-expressing cells. [Figure 34] 34A and 34B are graphs showing downregulation of expression of mast cell and melanocyte genes, respectively, associated with mast cell function in skin biopsies of cynomolgus macaques following dosing with mAb12. [Figure 35] Figures 35A and 35B are representative images of biopsy sections from animals treated with mAb12 before (Figure 35A) and at day 30 (Figure 35B). [Figure 36] Figures 36A and 36B are tables showing mast cell counts, with mean data shown in Figure 36A and aggregate data shown in Figure 36B. [Figure 37A] 37A, 37B, and 37C are graphs analyzing mean corpuscular hemoglobin (FIG. 37A), mean corpuscular hemoglobin concentration (FIG. 37B), and mean corpuscular volume (FIG. 37C). [Figure 37B] See legend to Figure 37A. [Figure 37C] See legend to Figure 37A. [Figure 38] 1 is a graph showing circulating levels of mAb12 from serum samples collected from monkeys over the course of the study. [Figure 39] 1 is a graph showing the presence of anti-drug antibodies (ADA) in monkeys over the course of the study (red line indicates the cut point of the assay). [Figure 40] Figures 40A-40F are graphs showing that administration of the bispecific antibody CDX-622 did not result in significant reductions in hematological parameters in a study in cynomolgus macaques. [Figure 41A] 41A-41F are graphs showing the decreased expression of several selected genes associated with mast cell function in skin biopsies of cynomolgus macaques following dosing with CDX-622. [Figure 41B] See legend to Figure 41A. [Figure 41C] See legend to Figure 41A. [Figure 41D] See legend to Figure 41A. [Figure 41E] See legend to Figure 41A. [Figure 41F] See legend to Figure 41A. [Figure 42] 1 is a graph showing inhibition of TSLP-induced CD80 expression on human dendritic cells by bispecific antibody CDX-622. [Figure 43] 1 is a graph showing inhibition of TSLP binding to TSLP-R by bispecific antibody CDX-622 (ELISA). [Figure 44] 1 is a graph showing inhibition of TSLP-mediated cell proliferation (BaF3 cells) by bispecific antibody CDX-622. [Figure 45] 1 is a graph showing the viability of human eosinophils treated with bispecific antibody CDX-622. [Figure 46] 1 is a graph showing SCF-induced cytokine release from human primary mast cells treated with antibody mAb12, antibody 1D10, or bispecific CDX-622. [Figure 47] 1 is a graph showing MCP-1 induction with SCF and TSLP in LAD2 cells. [Figure 48] 1 is a graph showing simultaneous blockade of SCF and TSLP in LAD2 cells treated with antibody mAb12, antibody 1D10, or bispecific CDX-622. [Figure 49] 1 is a graph showing inhibition of TSLP-induced CD80 expression on human dendritic cells treated with antibody 1D10 or bispecific CDX-622. DETAILED DESCRIPTION OF THE INVENTION

[0093] IV. DETAILED DESCRIPTION OF THE INVENTION In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0094] A. definition As used herein, the term "subject" includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat subjects with immune disorders. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0095] As used herein, the terms "binding domain" and "antigen-binding portion" are used interchangeably and refer to the portion of a protein or antibody that contains amino acid residues that interact with an antigen. Binding domains include, but are not limited to, antibodies (e.g., full-length antibodies) and their antigen-binding portions. A binding domain confers its specificity and affinity for an antigen to a binding agent. The term also encompasses any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources or may be partially or wholly synthetically produced.

[0096] The term "antibody" as referred to herein includes whole antibodies. In a preferred embodiment, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (referred to herein as V H Each light chain is composed of a light chain variable region (abbreviated as V) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. L The V domain consists of a V-chain constant region (CL) and a V-chain constant region (CL). H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0097] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment" or "binding domain"), as used herein, refers to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., human TSLP or human SCF). Such "fragments" are, for example, about 8 to about 1500 amino acids in length, preferably about 8 to about 745 amino acids in length, and preferably about 8 to about 300, e.g., about 8 to about 200 amino acids, or about 10 to about 50 or 100 amino acids in length. It has been shown that the antigen-binding function of an antibody can be accomplished by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include (i) V L Domain, V H (ii) a Fab fragment, which is a monovalent fragment consisting of the V, CL, and CH1 domains; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; H (iv) a Fd fragment consisting of a V domain and a CH1 domain; (iv) a V fragment consisting of a single arm of an antibody; L Domains and V H Fv fragment consisting of domains; (v) V H and (vi) an isolated complementarity-determining region (CDR), or (vii) a combination of two or more isolated CDRs, optionally joined by a synthetic linker. Additionally, the two domains of the Fv fragment, V, are included. L and V H Although encoded by separate genes, V L Area and V HUsing recombinant methods, they can be joined by synthetic linkers that allow them to be produced as a single protein chain in which the regions pair to form a monovalent molecule (known as single-chain Fvs (sFvs); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0098] The term "monoclonal antibody," as used herein, refers to an antibody that displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that displays a single binding specificity and has variable and optional constant regions derived from human germline immunoglobulin sequences. In one aspect, a human monoclonal antibody is produced by a hybridoma, which comprises a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome includes human heavy chain and light chain transgenes, and which is fused to an immortalized cell.

[0099] The term "recombinant human antibody," as used herein, includes all human antibodies prepared, expressed, created, or isolated by recombinant means, e.g., (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or from hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombinant combinatorial human antibody libraries, and (d) antibodies prepared, expressed, created, or isolated by any other means involving splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize particular human germline immunoglobulin sequences encoded by germline genes, but include subsequent rearrangements and mutations that occur, e.g., during antibody maturation. As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable region contains the antigen-binding domain, which is encoded by various genes that are rearranged to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by multiple single amino acid changes (referred to as somatic mutation or hypermutation) to increase the affinity of the antibody for the foreign antigen. The constant region further changes in response to the antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules encoding light and heavy chain immunoglobulin polypeptides in response to an antigen may not have sequence identity with the original nucleic acid molecule, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0100] The term "human antibody" includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. The human antibodies of the present invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation) (see Lonberg, N. et al. (1994) Nature 368(6474): 856-859; Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci 764:536-546). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., chimeric antibodies and humanized antibodies).

[0101] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds human TSLP or human SCF is substantially free of antibodies that specifically bind to antigens other than human TSLP or human SCF; an isolated antibody that specifically binds human TSLP is substantially free of antibodies that specifically bind to antigens other than human TSLP). However, an isolated antibody that specifically binds to an epitope may have cross-reactivity to the same antigen from a different species. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals.

[0102] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed both from contiguous amino acids or from noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody (i.e., epitope mapping) are well known in the art, and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or consecutive peptides from an antigen (e.g., TSLP or SCF) are tested for reactivity with a given antibody (e.g., anti-TSLP or anti-SCF antibody).Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)).

[0103] The term "an antibody that binds to the same epitope" as another antibody is intended to encompass antibodies that interact with, i.e., bind to, the same structural region on human TSLP or human SCF as a reference anti-TSLP antibody or a reference anti-SCF antibody. The "same epitope" to which the antibody binds can be a linear epitope or a conformational epitope formed by tertiary folding of the antigen.

[0104] The term "competing antibody" refers to an antibody that competes with a reference anti-TSLP antibody for binding to human TSLP or that competes with a reference anti-SCF antibody for binding to human SCF, i.e., an antibody that competitively inhibits the binding of a reference anti-TSLP antibody to TSLP or competitively inhibits the binding of a reference anti-SCF antibody to SCF. A "competing antibody" may bind to the same epitope on TSLP or SCF as the reference anti-TSLP or reference anti-SCF antibody, may bind to an overlapping epitope, or may sterically interfere with the binding of the reference anti-TSLP antibody to TSLP or the binding of the reference anti-SCF antibody to SCF.

[0105] Antibodies that recognize the same epitope or antibodies that compete for binding can be identified using routine techniques.Such techniques include, for example, immunoassays that show the ability of one antibody to block the binding of another antibody to a target antigen, i.e., competitive binding assays.Competitive binding is determined in assays in which the immunoglobulin under test inhibits the specific binding of a reference antibody to a common antigen, such as TSLP or SCF. Numerous types of competitive binding assays are available, such as solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct label RIAs using I-125 labels (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIAs (see Cheung et al., Virology 176:546 (1990); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, such assays involve the use of purified antigen or cells bearing either of these bound to a solid surface, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually, the test immunoglobulin is present in excess.Typically, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or greater than 75%.

[0106] Other techniques include epitope mapping methods, such as X-ray analysis of crystals of antigen:antibody complexes, which provide atomic resolution of epitopes. Other methods monitor the binding of antibodies to antigen fragments or mutated variants of antigens, where loss of binding due to alteration of amino acid residues in the antigen sequence is often considered to be an indication of epitope components. In addition, computational combinatorial methods for epitope mapping can also be used. These methods rely on the ability of a target antibody to affinity isolate specific short peptides from combinatorial phage display peptide libraries. These peptides are then used as clues to define the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have also been developed that have been shown to map conformationally discontinuous epitopes.

[0107] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to antibody binding to an epitope on a given antigen. Typically, an antibody binds to an epitope of approximately 10 as determined by surface plasmon resonance (SPR) technology on a BIACORE 2000 instrument (e.g., using recombinant human TSLP or recombinant human SCF as the analyte and the antibody as the ligand). -7 Less than M, e.g., about 10 -8 Under M, 10 -9 Less than M or 10 -10 The equilibrium dissociation constant (K D) and binds to a predetermined antigen with an affinity that is at least two-fold greater than its affinity to bind to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0108] As used herein, "K D The term "antibody-antigen interaction" is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the human antibodies of the invention have a dissociation equilibrium constant of approximately 10 as determined by surface plasmon resonance (SPR) technology on a BIACORE 2000 instrument (e.g., using recombinant human TSLP or recombinant human SCF as the analyte and the antibody as the ligand). -8 Less than M, e.g. 10 -9 Less than M or 10 -10 A dissociation equilibrium constant (K D ) binds to TSLP or SCF.

[0109] The term "kd," as used herein, is intended to refer to the off-rate constant for dissociation of an antibody from the antibody / antigen complex.

[0110] The term "ka," as used herein, is intended to refer to the on rate constant for the association of an antibody with an antigen.

[0111] The term "EC50," as used herein, refers to the concentration of an antibody or antigen-binding portion thereof that induces 50% of the maximal response, i.e., a response that is halfway between the maximal response and the baseline, in either an in vitro or in vivo assay.

[0112] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. In one embodiment, human monoclonal antibodies of the invention are of the IgG1 isotype. In another embodiment, human monoclonal antibodies of the invention are of the IgG2 isotype.

[0113] As used herein, the terms "inhibit" or "block" (e.g., with respect to inhibiting / blocking TSLP binding to the TSLP receptor (TSLP-R) and / or SCF binding to c-Kit) are used interchangeably and include both partial and complete inhibition / blocking. Inhibition / blocking preferably reduces or alters the normal level or type of activity that occurs when binding occurs without inhibition or blockage. Inhibition and blocking are also intended to include any measurable decrease in binding affinity of the TSLP-R when contacted with an anti-TSLP antibody compared to the TSLP-R not contacted with the anti-TSLP antibody, e.g., inhibiting CD70 binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. Similarly, inhibition and blocking are also intended to include any measurable decrease in the binding affinity of c-Kit when contacted with an anti-SCF antibody compared to c-Kit not contacted with the anti-SCF antibody, e.g., inhibiting CD70 binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In one embodiment, the anti-TSLP antibody (or anti-SCF antibody) inhibits TSLP-R (or c-Kit) binding by at least about 70%. In another embodiment, the anti-TSLP antibody (or anti-SCF antibody) inhibits TSLP-R (or c-Kit) binding by at least 80%. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of TSLP or SCF when contacted with an anti-TSLP (or anti-SCF) antibody compared to TSLP or SCF not contacted with the anti-TSLP (or anti-SCF) antibody, e.g., inhibiting TSLP or SCF binding by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.In one embodiment, the anti-TSLP antibody inhibits TSLP binding by at least about 70%. In another embodiment, the anti-TSLP antibody inhibits TSLP binding by at least 80%. In one embodiment, the anti-SCF antibody inhibits SCF binding by at least about 70%. In another embodiment, the anti-SCF antibody inhibits SCF binding by at least 80%.

[0114] The term "cross-reactive," as used herein, refers to the ability of an anti-TSLP-binding domain or anti-SCF-binding domain of the present invention to bind to TSLP or SCF, respectively, from different species. For example, a TSLP-binding domain of the present invention that binds to human TSLP may also bind to TSLP from another species. Similarly, an anti-SCF-binding domain of the present invention that binds to human SCF may also bind to SCF from another species. As used herein, cross-reactivity is measured by detecting specific reactivity with purified antigen in a binding assay (e.g., SPR, ELISA), or by binding to or otherwise functionally interacting with cells that physiologically express TSLP or SCF. Methods for determining cross-reactivity include standard binding assays described herein, such as Biacore™ surface plasmon resonance (SPR) analysis using a Biacore™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden), or by flow cytometry techniques.

[0115] As used herein, the term "naturally occurring," when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0116] The present invention also encompasses "conservative sequence modifications" of any of the sequences set forth in SEQ ID NOs: 1-349, i.e., modifications to the nucleotide and amino acid sequences that do not prevent the VH and VL sequences encoded by or containing the nucleotide or amino acid sequences from binding to antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. Modifications can be introduced into SEQ ID NOs: 1-349 by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an anti-TSLP or anti-SCF antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0117] In certain embodiments, conservative amino acid sequence modifications refer to at most 1, 2, 3, 4, or 5 conservative amino acid substitutions relative to the CDR sequences described herein. For example, each such CDR may contain up to 5 conservative amino acid substitutions, such as up to 4 (i.e., less than 4) conservative amino acid substitutions, such as up to 3 (i.e., less than 3) conservative amino acid substitutions, such as up to 2 (i.e., less than 2) conservative amino acid substitutions, or no more than 1 conservative amino acid substitution.

[0118] Alternatively, in another embodiment, mutations can be introduced randomly throughout all or part of the coding sequence of the anti-TSLP or anti-SCF binding domain, such as by saturation mutagenesis, and the resulting modified anti-TSLP or anti-SCF antibodies can be screened for binding activity.

[0119] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids, or given sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions to the complement of the strand.

[0120] With respect to amino acids, the term "substantial homology" indicates that two amino acid sequences, or sequences thereof, when optimally aligned and compared, are identical in at least about 80% of the amino acids, usually at least about 90% to 95%, and more preferably at least about 98% to 99% or 99.5% of the amino acids, with appropriate amino acid insertions or deletions.

[0121] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0122] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0123] The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences identical to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences identical to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov.

[0124] In a specific embodiment, an antibody or bispecific antibody described herein comprises a modified Fc region or domain, wherein the Fc region or domain comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue.

[0125] In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and contains at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or one of the following amino acid modifications: 234A, 234D, 234E, 234N, 234D, 234E, 234F, 234G, 234H, 234I, 234J, 234K ... 34Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 23 5F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R.243W, 243L243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 2 99V, 299H, 299F, 299E, 313F, 322Q, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 3 27N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 3 and at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue selected from the group consisting of 30K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A. Optionally, the Fc region or Fc domain may include additional and / or alternative non-naturally occurring amino acid residues known to those of skill in the art (e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; and WO05 / 040217). In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is a human IgG2 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue, which is equivalent to an amino acid residue described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG3 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue, which is equivalent to an amino acid residue described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG4 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue, which is equivalent to an amino acid residue described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art.

[0126] In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is a human IgG1 Fc region or domain and contains at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or one of the following amino acid modifications: 234A, 234D, 234E, 234N, 234D, 234E, 234F, 234G, 234H, 234I, 234J, 234K ... 34Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 23 5F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R.243W, 243L243Y, 243R, 243Q, 244H, 245A, 247V, 247G, 252Y, 254T, 256E, 2621, 262A, 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 269H, 269Y, 269F, 269R, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I, 299L, 299A, 299S, 2 99V, 299H, 299F, 299E, 313F, 322Q, 325Q, 325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 3 27N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 3 and at least one non-naturally occurring amino acid residue (e.g., 1, 2, 3, 4, 5, or 6) selected from the group consisting of 30K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, and 332A. Optionally, the Fc region or Fc domain may include additional and / or alternative non-naturally occurring amino acid residues known to those of skill in the art (e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO 01 / 58957; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; and WO05 / 040217). In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is a human IgG2 Fc region or domain and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue, which is equivalent to an amino acid residue described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG3 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue, which is equivalent to an amino acid residue described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art. In a specific embodiment, the antibodies described herein comprise a modified Fc region or domain, wherein the Fc region or domain is that of a human IgG4 and comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) amino acid modification (e.g., substitution, deletion, or addition) or at least one (e.g., 1, 2, 3, 4, 5, or 6) non-naturally occurring amino acid residue, which is equivalent to an amino acid residue described herein for a human IgG1 Fc region or domain, as can be determined by one of skill in the art.

[0127] In certain aspects, provided herein is an antibody comprising an Fc region or Fc domain, wherein the Fc region or Fc domain is the Fc region or Fc domain of a human IgG1 and comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330, and 332, as numbered according to the EU index as set forth in Kabat. In a specific embodiment, provided herein is an antibody comprising an Fc region or Fc domain, wherein the Fc region or Fc domain is the Fc region or Fc domain of a human IgG1 and comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L, and 332E, as numbered according to the EU index as set forth in Kabat. Optionally, the Fc region or Fc domain may further comprise an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered according to the EU index as set forth in Kabat. In a specific aspect, provided herein is an antibody comprising an Fc region or Fc domain, wherein the Fc region or Fc domain is that of a human IgG1 and comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L, and 332E, as numbered according to the EU index as set forth in Kabat, and wherein at least one non-naturally occurring amino acid at one or more positions is selected from the group consisting of 252Y, 254T, and 256E, as numbered according to the EU index as set forth in Kabat. In a specific aspect, provided herein is an antibody comprising an Fc region or Fc domain, wherein the Fc region or Fc domain is that of a human IgG2, human IgG3, or human IgG4 and comprises at least one non-naturally occurring amino acid residue that is the equivalent, as can be determined by one of skill in the art, to an amino acid residue described herein for the human IgG1 Fc region or human IgG1 Fc domain.In a specific embodiment, provided herein is an antibody comprising an Fc region or Fc domain, wherein the Fc region or Fc domain is an Fc region or Fc domain of human IgG2, human IgG3, or human IgG4, and comprises at least one non-naturally occurring amino acid residue at one or more positions that are equivalent to the positions described herein for a human IgG1 Fc region or human IgG1 Fc domain, as can be determined by one of skill in the art. In one embodiment, an Fc region or Fc domain comprising such a sequence exhibits one or more Fc activities, e.g., binding affinity for an Fc receptor, or an effector function such as ADCC or CDC. In a specific embodiment, an Fc region or Fc domain comprising such a sequence exhibits reduced Fc activity, e.g., reduced binding affinity for an Fc receptor, or reduced effector function such as ADCC or CDC. In a particular embodiment, an Fc region or Fc domain comprising such a sequence exhibits improved FcRn activity, e.g., improved half-life.

[0128] Ghetie et al., 1997, Nat Biotech.15:637-40;Duncan et al., 1988, Nature 332:563-564;Lund et al., 1991, J. Immunol 147:2657-2662;Lund et al., 1992, Mol Immunol 29:53-59;Alegre et al., 1994, Transplantation 57:1537-1543;Hutchins et al., 1995, Proc Natl. Acad Sci USA 92: 11980-11984; Jefferis et al., 1995, Immunol Lett. 44: 111-117; Lund et al., 1995, Faseb J 9: 115-119; 1996, J Immunol 157:4963-4969; Armor et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164: 1925-1933; Xu et al., 2000, Cell Immunol 200: 16-26; Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490); U.S. Patent No. 5,624,821; No. 5,885,573; No. 5,677,425; No. 6,165,745; No. 9,046; No. 6,121,022; No. 5,624,821; No. 5,648,260; No. 6,528,624; No. 6,194,551; No. 6,737,056; No. 6 ,821,505; 6,277,375; 8,163,882; 7,355,008; 7,960,512; 8,039,592; 8,039,359; 8,101,720; 7,214,775; 7,682,610; 7,741,442; U.S. Patent Application Publication No. 2004 / 0002587 and PCT Publication Nos. WO 94 / 29351; WO 99 / 58572; WO 00 / 42072; WO 04 / 029207; WO 04 / 099249; and WO 04 / 063351.

[0129] In specific embodiments, the antibodies described herein comprise a modified (e.g., mutated) human IgG1 Fc region or domain comprising the non-naturally occurring amino acids 234A, 235Q, and 322Q, as numbered by the EU index as set forth in Kabat. In certain embodiments, the modified (e.g., mutated) human IgG1 Fc region or domain further comprises the non-naturally occurring amino acids 252Y, 254T, and 256E, as numbered by the EU index as set forth in Kabat.

[0130] In certain embodiments, the antibodies described herein comprise an altered (e.g., mutated) human IgG2 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalent, as can be determined by one of skill in the art, to 234A, 235Q, and 322Q for the human IgG1 Fc region or domain as numbered by the EU index as set forth in Kabat. In certain embodiments, the antibodies described herein comprise an altered (e.g., mutated) human IgG2 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalent, as can be determined by one of skill in the art, to 234A, 235Q, 322Q, 252Y, 254T, and 256E for the human IgG1 Fc region or domain as numbered by the EU index as set forth in Kabat.

[0131] In certain embodiments, the antibodies described herein comprise an altered (e.g., mutated) human IgG3 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalent, as can be determined by one of skill in the art, to 234A, 235Q, and 322Q for the human IgG1 Fc region or domain as numbered by the EU index as set forth in Kabat. In certain embodiments, the antibodies described herein comprise an altered (e.g., mutated) human IgG3 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalent, as can be determined by one of skill in the art, to 234A, 235Q, 322Q, 252Y, 254T, and 256E for the human IgG1 Fc region or domain as numbered by the EU index as set forth in Kabat.

[0132] In certain embodiments, the antibodies described herein comprise an altered (e.g., mutated) human IgG4 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalent, as can be determined by one of skill in the art, to 234A, 235Q, and 322Q for the human IgG1 Fc region or domain as numbered by the EU index as set forth in Kabat. In certain embodiments, the antibodies described herein comprise an altered (e.g., mutated) human IgG4 Fc region or domain, which comprises non-naturally occurring amino acids that are equivalent, as can be determined by one of skill in the art, to 234A, 235Q, 322Q, 252Y, 254T, and 256E for the human IgG1 Fc region or domain as numbered by the EU index as set forth in Kabat.

[0133] In a specific embodiment, the antibodies described herein comprise the VL and VH CDR sequences set forth herein and a modified (e.g., mutated) human IgG1 Fc region or domain, wherein the modified (e.g., mutated) human IgG1 Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q, and 322Q as numbered by the EU index as set forth in Kabat.

[0134] In a preferred embodiment, the antibodies described herein comprise the VL and VH CDR sequences set forth herein and a modified (e.g., mutated) human IgG1 Fc region or domain, wherein the modified (e.g., mutated) human IgG1 Fc region or domain comprises the non-naturally occurring amino acids 234A, 235Q, 322Q, 252Y, 254T, and 256E, as numbered by the EU index as set forth in Kabat.

[0135] B. Anti-TSLP antibody Provided herein are novel anti-TSLP antibodies and their binding domains. The term "TSLP" (also referred to as "thymic stromal lymphopoietin") refers to a cytokine involved in the maturation of T cell populations through activation of antigen-presenting cells. TSLP is produced by non-hematopoietic cells, such as fibroblasts, epithelial cells, and various types of stromal or stromal-like cells. TSLP signals through the TSLP receptor (TSLPR, also referred to as "CRFL2"). TSLPR forms a functional complex with TSLP and IL7R and stimulates cell proliferation by activating STAT3 and STAT5, as well as JAK2. The TSLPR chain is closely related to the common receptor gamma chain, which is expressed on a wide range of cell types in the adaptive and innate immune systems and is associated with hematopoietic development.

[0136] TSLP also influences dendritic cell polarization, triggering the production of T helper (Th) 2 cytokines, directly promoting T cell proliferation in response to T cell receptor activation and Th2 cytokine production, and supporting the expansion and differentiation of B cells. TSLP also amplifies Th2 cytokine production by mast cells and natural killer T cells. These properties give TSLP a crucial role in initiating Th2-mediated inflammation. TSLP expression has therefore been linked to many pathologies, including asthma, inflammatory arthritis, atopic dermatitis, eczema, eosinophilic esophagitis, and other conditions.

[0137] The term "TSLP" includes any variant or isoform of TSLP that is naturally expressed by a cell (e.g., human TSLP deposited with the UniProt® Consortium with accession number Q969D9-1, as set forth in SEQ ID NO:70).

[0138] Thus, the TSLP antibody (or binding domain) of the present invention may cross-react with TSLP from species other than humans. Alternatively, the TSLP antibody (or binding domain) may be specific for human TSLP and may not exhibit any cross-reactivity with other species. TSLP, or any variants and isoforms thereof, may either be isolated from cells or tissues that naturally express them, or may be recombinantly produced using techniques well known in the art and / or described herein. Preferably, the TSLP antibody (or binding domain) targets human TSLP with a normal glycosylation pattern.

[0139] In another embodiment, the anti-TSLP antibody (or binding domain) comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region of any one of antibodies 1D10-A, 1D10-B, 1D10-C, 1D10-D, 1D10-E, 1D10-F, 1D10-G, 1D10-H, or 1D10-I (as shown in tail 5 of Example 2). For example, the anti-TSLP antibody or binding domain thereof comprises the CDR1, CDR2, and CDR3 domains of the heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:1, 5, 9, or 13, and / or the CDR1, CDR2, and CDR3 domains of the light chain variable region having the amino acid sequence set forth in SEQ ID NO:17, 21, 25, or 29. In another embodiment, the anti-TSLP antibody (or binding domain) comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences, or conservative sequence modifications thereof, respectively, set forth in Table 5A, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences, or conservative sequence modifications thereof, respectively, set forth in Table 5B. In another embodiment, the anti-TSLP antibody or (binding domain) comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:2, 3, and 4, (b) SEQ ID NOs:6, 7, and 8, (c) SEQ ID NOs:10, 11, and 12, and (d) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and / or light chain CDR1, CDR2, and CDR3 domains having the sequences set forth in (a) SEQ ID NOs:18, 19, and 20, (b) SEQ ID NOs:22, 23, and 24, (c) SEQ ID NOs:26, 27, and 28, and (d) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof, as set forth in Table 1A. In one embodiment, the anti-TSLP antibody (or binding domain) comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:6, 7, and 8, and the light chain CDR1, CDR3, and CDR3 sequences set forth in SEQ ID NOs:18, 19, and 20, respectively.

[0140] In another embodiment, the anti-TSLP antibody (or binding domain) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, or 13. In another embodiment, the antibody or binding domain thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, 21, 25, or 29. In another embodiment, the antibody (or binding domain) comprises a combination of heavy and / or light chain variable regions having the amino acid sequences set forth in Table 1B. In yet another embodiment, the anti-TSLP antibody (or binding domain) comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 4. In one embodiment, the anti-TSLP antibody (or binding domain) comprises the heavy and light chain variable region sequences set forth in SEQ ID NO: 5 and 17, respectively.

[0141] Also provided are sequences substantially identical to the anti-TSLP antibodies (or binding domains) described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (identical) to the foregoing sequences). In one embodiment, the anti-TSLP antibody (or binding domain) comprises a heavy chain variable region comprising a sequence set forth in Table 1B or Table 4, or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0142] Anti-TSLP antibodies (and binding domains thereof) that compete for binding with or bind to the same epitope as any of the anti-TSLP antibodies (or binding domains) described herein are also suitable for use and are provided herein. For example, in one embodiment, an anti-TSLP antibody (or binding domain) competes for binding to TSLP with antibody 1D10 described herein (or an antibody having heavy chain CDRs and / or light chain CDRs and / or heavy chain variable region sequences corresponding to antibody 1D10 described herein). In another embodiment, the antibody or anti-TSLP binding domain thereof binds to the same epitope on TSLP as antibody 1D10 described herein (or an antibody having heavy chain CDRs and / or heavy chain variable region sequences corresponding to antibody 1D10 described herein).

[0143] C. Anti-SCF antibody / binding domain As used herein, the terms "stem cell factor," "SCF," "mast cell growth factor," "MGF," "KIT ligand," "KL," and "steel factor" are used interchangeably and include variants, isoforms, species homologs, and analogs of human SCF that share at least one epitope with SCF. The complete sequence of SCF can be found under UniProt accession number P21583, as set forth in SEQ ID NO:71.

[0144] SCF is a ligand for the receptor protein tyrosine kinase KIT ("c-Kit") and is involved in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, gametogenesis, mast cell development, migration, survival, and activation, and melanogenesis. Upon binding to c-Kit, SCF activates several signaling pathways, promoting phosphorylation of PIK3R1, a regulatory subunit of phosphatidylinositol 3-kinase, and subsequently activating the kinase AKT1. SCF binding to c-Kit also signals via GRB2, activating RAS, RAF1, and the MAP kinases MAPK1 / ERK2 and / or MAPK3 / ERK1. SCF binding also promotes activation of STAT family members STAT1, STAT3, and STAT5, as well as PLCG1, leading to the production of the cell signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate. SCF / c-Kit binding also acts synergistically with other cytokines such as interleukins.

[0145] In one embodiment, the anti-SCF antibody (or binding domain thereof) comprises the heavy and light chain CDRs or variable regions of any one of anti-SCF antibodies mAb12-A, mAb12-B, mAb12-C, mAb12-D, mAb12-E, mAb12-F, mAb12-G, mAb12-H, or mAb12-I (as set forth in Table 9 in Example 9). In another embodiment, the anti-SCF antibody (or binding domain) comprises the CDR1, CDR2, and CDR3 domains of a heavy chain variable region having the sequence set forth in SEQ ID NO:33, 37, 41, or 45, and / or the CDR1, CDR2, and CDR3 domains of a light chain variable region having the sequence set forth in SEQ ID NO:49, 53, 57, or 61. In another embodiment, the anti-SCF antibody (or binding domain) comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 8A or conservative sequence modifications thereof, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in Table 8B or conservative sequence modifications thereof. In another embodiment, the anti-SCF antibody (or binding domain) comprises heavy chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:34, 35, and 36, (b) SEQ ID NOs:38, 39, and 40, (c) SEQ ID NOs:42, 43, and 44, and (d) SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and / or light chain CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (a) SEQ ID NOs:50, 51, and 52, (b) SEQ ID NOs:54, 55, and 56, (c) SEQ ID NOs:58, 59, and 60, and (d) SEQ ID NOs:62, 63, and 64, respectively, or conservative sequence modifications thereof, as set forth in Table 2A. In one embodiment, the anti-SCF antibody (or binding domain) comprises the heavy chain CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NOs:46, 47, and 48, and the light chain CDR1, CDR3, and CDR3 sequences set forth in SEQ ID NOs:58, 59, and 60, respectively.

[0146] In another embodiment, the anti-SCF antibody (or binding domain) comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:33, 37, 41, or 45. In another embodiment, the anti-SCF antibody (or binding domain) comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO:49, 53, 57, or 61. In another embodiment, the anti-SCF antibody (or binding domain) comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 2B. In yet another embodiment, the antibody (or binding domain) comprises a combination of heavy and light chain variable regions having the amino acid sequences set forth in Table 5 of Example 9. In one embodiment, the anti-SCF antibody (or binding domain) comprises the heavy and light chain variable region sequences set forth in SEQ ID NO:45 and 57, respectively.

[0147] Also provided are sequences substantially identical to the anti-SCF antibodies (or binding domains) described herein (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical (identical) to the foregoing sequences). In one embodiment, the anti-SCF antibody (or binding domain) comprises a heavy chain variable region comprising a sequence set forth in Table 2B or Table 5, or at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0148] Anti-SCF antibodies and their binding domains that compete for binding with any of the anti-SCF antibodies (or their binding domains) described herein or that bind to the same epitope as any of the anti-SCF antibodies (or their binding domains) described herein are also suitable for use and are provided herein. For example, in one embodiment, an anti-SCF antibody or its binding domain competes for binding to SCF with the antibody mAb12 described herein (or an antibody having heavy chain CDRs and light chain CDRs and / or heavy chain variable region sequences corresponding to the antibody mAb12 described herein). In another embodiment, the anti-SCF antibody (or binding domain) binds to the same epitope on SCF as the antibody mAb12 described herein (or an antibody having heavy chain CDRs and light chain CDRs and / or heavy chain variable region sequences corresponding to the antibody mAb12).

[0149] D. Bispecific and Multispecific Constructs Provided herein are bispecific constructs comprising an anti-TSLP antibody (or binding domain) or an anti-SCF antibody (or binding domain) linked to a second binding agent (e.g., a ligand, antibody, or antigen-binding portion thereof). Also provided are bispecific constructs comprising an anti-TSLP antibody (or binding domain) linked to an anti-SCF antibody (or binding domain). The term "bispecific construct," as used herein, also refers to a bispecific construct that is linked to one or more additional binding agents (i.e., a third, fourth, or fifth binding agent) to form a "multispecific construct."

[0150] A "bispecific" or "bifunctional" construct is an artificial hybrid having two different binding domain (e.g., heavy / light chain) pairs and two different binding sites. Bispecific constructs can be produced by a variety of methods, including hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). A bispecific construct having more than two different binding domains (e.g., three, four, or more) is a multispecific construct.

[0151] As used herein, the term "linked" refers to the association of two or more molecules. Linkage can be covalent or non-covalent. Linkage can also be genetic (i.e., recombinant fusion). Such linkage can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0152] For chemical conjugation, suitable reagents and methods for coupling two or more moieties, particularly two or more antibodies or their fragments, together are known in the art. A variety of coupling or cross-linking agents are commercially available and can be used to conjugate the anti-TSLP binding domain and the anti-SCF binding domain. Non-limiting examples include sulfo-SMCC, protein A, carboimide, dimaleimide, dithio-bis-nitrobenzoic acid (DTNB), and N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Sulfo-SMCC, SPDP, and DTNB are preferred agents, with sulfo-SMCC being particularly preferred. Other suitable procedures for cross-linking components (e.g., binding domains) using cross-linking agents are known in the art. See, e.g., Karpovsky, B. et al., (1984) J. Exp. Med. 160:1686; Liu, MA et al., (1985) Proc. Natl. Acad. Sci USA 82:8648; Segal, DM and Perez, P., U.S. Pat. No. 4,676,980; and Brennan, M. (1986) Biotechniques 4:424.

[0153] For genetic engineering, a nucleic acid molecule encoding an anti-TSLP antibody (or binding domain) or an anti-SCF antibody (or binding domain) can be inserted into an appropriate expression vector using standard recombinant DNA techniques. A nucleic acid molecule encoding a second binding agent (e.g., a ligand, antibody, or binding domain thereof) can also be inserted into the same expression vector such that it is operably linked (e.g., in-frame cloned) to the anti-TSLP antibody (or binding domain) or anti-SCF binding antibody (or binding domain), thereby resulting in an expression vector encoding a fusion protein that is a bispecific construct.

[0154] Exemplary bispecific constructs include combinations of binding agents that bind to targets listed in Table 3A, such as an anti-TSLP antibody (or binding domain) or an anti-SCF antibody (or binding domain) linked to a second binding agent that binds to a member of the TNF superfamily (e.g., TNFα), a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4), an interleukin (e.g., IL-23, IL-17A, or IL-13), an immunoglobulin (e.g., IgE), or an integrin (e.g., integrin alpha 4 beta 7). In one embodiment, the bispecific construct comprises an anti-TSLP antibody and an anti-SCF antibody (or antigen-binding domain) described herein having the heavy and light chain CDR sequences and / or full-length variable region sequences set forth in Tables 1A, 1B, and 2A, 2B, respectively.

[0155] Another exemplary bispecific construct comprises an anti-TSLP antibody (or binding domain) linked to an anti-SCF antibody (or binding domain), e.g., anti-TSLP antibody (or binding domain) 1D10-D linked to anti-SCF antibody (or binding domain) mAb12-G. Preferably, the anti-SCF binding moiety is operably linked to the C-terminal region of the heavy chain of the anti-TSLP binding moiety. Other suitable expression vectors and cloning strategies for preparing the bispecific constructs described herein are known in the art.

[0156] (Table 3A) TIFF2026508389000005.tif83140

[0157] Binding agents for use with the bispecific constructs include, for example, the antibodies (or binding domains thereof) listed in Table 3B.

[0158] Table 3B: Representative binding antibodies and their antigen-binding portions TIFF2026508389000006.tif230140

[0159] For expression of bispecific constructs in host cells, the coding regions of the binding moieties (e.g., antibodies or their binding domains) are combined with cloned promoter, leader sequence, translation initiation, leader sequence, constant region, 3' untranslated, polyadenylation, and transcription termination sequences to form an expression vector construct. These constructs can be used to express, for example, full-length human IgG1κ or human IgG4κ antibodies. Fully human, humanized, and chimeric antibodies used in the bispecific constructs described herein also include IgG2, IgG3, IgE, IgA, IgM, and IgD antibodies. Similar plasmids can be constructed for the expression of other heavy chain isotypes or for the expression of antibodies containing lambda light chains.

[0160] After preparing an expression vector encoding the bispecific construct, the bispecific construct can be recombinantly expressed in host cells using standard transfection methods. For example, in one embodiment, the nucleic acid encoding the bispecific construct can be ligated into an expression vector, such as a eukaryotic expression plasmid, such as those used by the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338841, or other expression systems known in the art. The purified plasmid carrying the cloned bispecific construct gene can be introduced into eukaryotic host cells, such as CHO cells or NSO cells, or alternatively, other eukaryotic cells, such as plant-derived cells, fungi, or yeast cells. The method used to introduce these genes can be one described in the art, such as electroporation, lipofectin, lipofectamine, or others. After introducing the expression vector into the host cells, cells expressing the bispecific construct can be identified and selected. These cells represent transfectomas, which can then be amplified and upscaled for their expression levels to generate bispecific constructs. Alternatively, these cloned bispecific constructs can be expressed in other expression systems, such as E. coli, or in whole organisms, or can be expressed synthetically. Recombinant bispecific constructs can be isolated and purified from these culture supernatants and / or cells.

[0161] The bispecific construct of the present invention, whether prepared by chemical conjugation or genetic engineering, can be isolated and purified using one or more well-established protein purification methods in the art.Preferred isolation and purification methods include, but are not limited to, gel filtration chromatography, affinity chromatography, anion exchange chromatography, and the like.A particularly preferred method is gel filtration chromatography, for example, using a Superdex 200 column.The isolated and purified bispecific construct can be evaluated using standard methods such as SDS-PAGE analysis.

[0162] Thus, in one embodiment, the anti-TSLP antibody (or binding domain) or the anti-SCF antibody (or binding domain) is genetically fused to an additional binding agent. Alternatively, the anti-TSLP antibody (or binding domain) and the anti-SCF antibody (or binding domain) are genetically fused to each other. In another embodiment, the anti-TSLP antibody (or binding domain) or the anti-SCF antibody (or binding domain) is chemically conjugated. Alternatively, the anti-TSLP antibody (or binding domain) and the anti-SCF antibody (or binding domain) are chemically conjugated to each other. In one embodiment, either one of the binding moieties further comprises a human IgG1 constant domain.

[0163] Bispecific constructs comprising an anti-TSLP antibody (or binding domain) linked to an anti-SCF antibody (or binding domain) include the following embodiments: (a) the anti-SCF binding moiety is linked to the C-terminus of the heavy chain of the anti-TSLP binding moiety; (b) the anti-SCF binding moiety is an scFv; (c) the anti-SCF binding moiety further comprises a human IgG1 constant domain; (d) the anti-TSLP binding moiety is linked to the C-terminus of the heavy chain of the anti-SCF binding moiety; or (e) the anti-TSLP binding moiety is an scFv.

[0164] Exemplary bispecific constructs include any one of the anti-TSLP antibodies (or binding domains) described herein having the CDRs or full-length heavy and light chain variable region sequences set forth in Tables 1A and 1B, or any one of the anti-SCF antibodies (or binding domains) described herein having the CDRs or full-length heavy and light chain variable region sequences set forth in Tables 2A and 2B, linked to a second binding domain (e.g., a ligand, antibody, or antigen-binding portion thereof). Exemplary bispecific constructs also include any one of the anti-TSLP antibodies (or binding domains) described herein having the CDRs or full-length heavy and light chain variable region sequences set forth in Tables 1A and 1B linked to any one of the anti-SCF antibodies (or binding domains) described herein having the CDRs or full-length heavy and light chain variable region sequences set forth in Tables 2A and 2B.

[0165] Other exemplary bispecific constructs include a combination of any one of the anti-TSLP antibodies (or antigen-binding fragments thereof) of Table 1A or 1B with any one of the anti-SCF antibodies (or antigen-binding fragments thereof) of Table 2A or 2B, where the bispecific construct comprises an anti-TSLP antibody linked to an anti-SCF scFv, and where the anti-TSLP antibody further comprises a human IgG1 constant domain. Alternatively, a bispecific construct includes a combination of an anti-TSLP antibody (or antigen-binding fragment thereof) of Table 1A or 1B combined with an anti-SCF antibody (or antigen-binding fragment thereof) of Table 2A or 2B, where the bispecific construct comprises an anti-SCF antibody linked to an anti-TSLP scFv, and where the anti-SCF antibody further comprises a human IgG1 constant domain.

[0166] Other exemplary bispecific constructs include an anti-TSLP binding domain and an anti-SCF binding domain comprising the amino acid sequences set forth in Table 10 of Example 19.

[0167] E. composition Also provided herein are compositions, e.g., compositions comprising any one or combination of antibodies (or binding domains) or bispecific constructs described herein, formulated together with a carrier (e.g., a pharmaceutically acceptable carrier).

[0168] As used herein, the terms "carrier" and "pharmaceutically acceptable carrier" include any and all physiologically compatible solvents, salts, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous administration (e.g., by injection or infusion), intramuscular administration, subcutaneous administration, parenteral administration, spinal administration, or epidermal administration. Depending on the route of administration, the active compound (i.e., any antibody (or binding domain) or bispecific construct described herein) may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0169] Examples of adjuvants that may be used with the antibodies (or binding domains), bispecific constructs described herein include, but are not limited to, Freund's incomplete and complete adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, Pa.); aluminum salts, such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron, or zinc; insoluble suspensions of acylated tyrosine; acylated sugars; cationic or anionic derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; cytokines, such as GM-CSF, interleukin-2, interleukin-7, interleukin-12, and other similar factors; 3D-MPL; CpG oligonucleotides; and monophosphoryl lipid A, e.g., 3-de-O-acylated monophosphoryl lipid A.

[0170] MPL adjuvant can be obtained from Corixa Corporation (Seattle, Wash.; see, for example, U.S. Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).CpG-containing oligonucleotides (wherein CpG dinucleotides are not methylated) are well known and are described, for example, in WO 96 / 02555, WO 99 / 33488, and U.S. Patent Nos. 6,008,200 and 5,856,462.Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.

[0171] Further alternative adjuvants include, for example, saponins such as Quil A or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, Mass.); escin; digitonin; or saponins from Gypsophila or quinoa; Montanide ISA 720 (Seppic, France); SAF (Chiron, California, United States); ISCOMS (CSL), MF-59 (Chiron); SBAS series adjuvants (e.g., SBAS-2 or SBAS-4 available from SmithKline Beecham, Rixensart, Belgium); Detox (Enhanzyn™) (Corixa, Hamilton, Mont.); RC-529 (Corixa, Hamilton, Mont.), and other aminoalkyl glucosaminide 4-phosphates (AGPs); WO polyoxyethylene ether adjuvants such as those described in J. Immunol. 99 / 52549A1; synthetic imidazoquinolines such as imiquimod [S-26308, R-837] (Harrison, et al., Vaccine 19: 1820-1826, 2001; and resiquimod [S-28463, R-848] (Vasilakos, et al., Cellular immunology 204: 64-74, 2000); Schiff bases of carbonyls and amines constitutively expressed on the surface of antigen-presenting cells and T cells, such as tucaresol (Rhodes, J. et al., Nature 377: 71-75, 1995); cytokines, chemokines, and costimulatory molecules, either as proteins or peptides (e.g., proinflammatory cytokines such as interferon, GM-CSF, IL-1α, IL-1beta, TGF-α, and TGF-β; Th1 inducers such as interferon gamma, IL-2, IL-12, IL-15, IL-18, and IL-21; Th2 inducers such as IL-4, IL-5, IL-6, IL-10, and IL-13; and other chemokines and costimulatory genes such as MCP-1, MIP-1α, MIP-1beta, RANTES, TCA-3, CD80, CD86, and CD40L); immunostimulatory agents that target ligands such as CTLA-4 and L-selectin, apoptosis-stimulating proteins and peptides such as Fas; vaxfectin (Reyes et al., Vaccine 19: 3778-3786, 2001) synthetic lipid-based adjuvants such as squalene, α-tocopherol, polysorbate 80, DOPC, and cholesterol; endotoxin, [LPS] (Beutler, B., Current Opinion in Microbiology 3: 23-30, 2000); ligands that trigger Toll receptors to produce Th1-inducing cytokines, such as synthetic mycobacterial lipoproteins, mycobacterial protein p19, peptidoglycan, teichoic acid, and lipid A; and CT (cholera toxin, subunits A and B) and LT (heat-labile enterotoxin from Escherichia coli, subunits A and B), a family of heat shock proteins (HSPs), and LLO (listeriolysin O; WO 01 / 72329). These and various additional Toll-like receptor (TLR) agonists are described, for example, in Kanzler et al., Nature Medicine, May 2007, Vol. 13, No. 5.

[0172] A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, as well as those derived from non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0173] The composition of the present invention can be administered by various methods known in the art.As those skilled in the art will understand, the route and / or mode of administration will vary depending on the desired results.The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.Many methods for preparing such formulations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0174] For the administration of the compound of the present invention by certain administration routes, it may be necessary to coat the compound with a material to prevent its inactivation, or to co-administer the compound with the material.For example, the compound can be administered to a subject in a suitable carrier, such as liposome or diluent.Acceptable diluents include physiological saline and aqueous buffer solution.Liposomes include not only conventional liposomes, but also water-in-oil-in-water CGF emulsions (Strejan et al. (1984) J.Neuroimmunol.7:27).

[0175] Carrier includes sterile aqueous solution or sterile dispersion and sterile powder for preparing sterile injectable solution or sterile injectable dispersion when used.The use of such media and agent for pharmaceutical active substance is known in the art.Except that any conventional media or agent is incompatible with active compound, it is contemplated to use it in the pharmaceutical composition of the present invention.Auxiliary active compounds can also be incorporated into the composition.

[0176] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0177] Sterile injectable solution can be prepared by incorporating active compound into appropriate solvent with one or combination of above-listed components as needed, and then carry out sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains base dispersion medium and other components that are required from above-listed components.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which makes the powder of active ingredient and any additional required components from its solution that has been previously sterilized and filtered.

[0178] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, antibodies of the invention may be administered by subcutaneous or intramuscular injection once or twice weekly, or once or twice monthly.

[0179] For the sake of ease of administration and uniformity of dosage, it is particularly advantageous to prepare parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit that is suitable as a unitary dosage for the subject to be treated; each unit contains a predetermined amount of active compound that is calculated to produce desired therapeutic effect together with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on (a) the unique characteristics of active compound and the specific therapeutic effect that should be achieved, and (b) the constraints inherent in the technical field of compounding this active compound for treating the sensitivity of individual.

[0180] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0181] For therapeutic compositions, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be provided in unit dosage form and may be prepared by any method known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 0.001 percent to about 90 percent of the active ingredient, preferably from about 0.005 percent to about 70 percent, and most preferably from about 0.01 percent to about 30 percent.

[0182] The preparation of the present invention suitable for vaginal administration also includes pessaries, tampons, creams, gels, pastes, foams or spray formulations containing carriers known to be suitable in the art.The dosage forms for topical or transdermal administration of the composition of the present invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

[0183] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral administration and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0184] Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0185] These compositions may also contain auxiliary substances such as preservatives, wetting agents, emulsifiers, and dispersants.Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like.It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like, in the composition.In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0186] When the compounds of the present invention are administered to humans and animals as pharmaceuticals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably 0.005 to 70%, e.g., 0.01 to 30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0187] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0188] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective for achieving the desired therapeutic response without causing toxicity to the patient for a particular patient, composition, and administration method. The selected dosage level will vary depending on various pharmacokinetic factors, including the activity of the particular composition of the present invention or its ester, salt, or amide employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field. A physician or veterinarian with ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian can start the dose of the compound of the present invention employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of the composition of the present invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such effective dose generally depends on the above factors. Administration is preferably intravenous, intramuscular, intraperitoneal, or subcutaneous, and is preferably administered close to the target site. If desired, the effective daily dose of the therapeutic composition can be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. Although the compound of the present invention can be administered alone, it is preferred to administer the compound as a pharmaceutical formulation (composition).

[0189] Therapeutic compositions can be administered using medical devices known in the art. For example, in a preferred embodiment, the therapeutic compositions of the present invention can be administered using a needleless hypodermic injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for delivering medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses an implantable variable flow rate infusion device for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0190] In certain embodiments, antibodies (or binding domains) or bispecific constructs of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported to designated cells or organs, thereby improving targeted drug delivery (see, e.g., VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species of which may constitute components of the formulations and molecules of the present invention; p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); also see K. Keinanen; ML See also Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes contain a targeting moiety.In the most preferred embodiment, the therapeutic compound in the liposome is delivered by bolus injection to a site adjacent to the tumor or infection. The composition must be fluid enough to allow easy syringability. The composition must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[0191] The ability of a compound to inhibit inflammation can be evaluated in an animal model system that predicts its effectiveness in human asthma patients.Alternatively, this property of a composition can be evaluated by examining the inhibitory ability of a compound, such as in vitro inhibition, by assays known to those skilled in the art.A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate symptoms in a subject.Those skilled in the art will be able to determine such amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0192] The composition must be sterile and fluid enough to allow delivery by syringe. In addition to water, the carrier can be isotonic buffered saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. In many cases, it is preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including an agent that delays absorption, such as aluminum monostearate or gelatin in the composition.

[0193] When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

[0194] F. nucleic acid The term "nucleic acid molecule," as used herein, is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, but preferably are double-stranded DNA.

[0195] The term "isolated nucleic acid molecule" refers to an antibody (or binding domain, e.g., V) that binds TSLP and / or SCF. H , V L As used herein with reference to nucleic acids encoding the CDRs (CDR3), as well as bispecific constructs comprising such antibodies, the term is intended to refer to nucleic acid molecules in which the nucleotide sequence encoding the antibody (or binding domain) or bispecific is free of other nucleotide sequences encoding the antibody (or binding domain) or bispecific, such as other sequences that may naturally flank the nucleic acid in human genomic DNA. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0196] The nucleic acid molecules of the present invention, whether from cDNA, genomic, or a mixture thereof, are often native sequences (except for modified restriction sites and the like), but can be mutated according to standard techniques to provide gene sequences. In the case of coding sequences, these mutations can affect the amino acid sequence as desired. In particular, DNA sequences that are substantially identical to or derived from the native V, D, J, constant, switch, and other such sequences described herein are contemplated (where "derived" indicates that the sequence is identical to or modified from another sequence).

[0197] A nucleic acid is "operably linked" or "operatively linked" when it is placed into a functional relationship with another nucleic acid sequence. By way of example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. With respect to transcriptional regulatory sequences, operably linked means that the DNA sequences being linked are contiguous, and, where necessary to join two protein-coding regions, contiguous and in reading frame. In the case of switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

[0198] Also provided are isolated nucleic acid molecules encoding the antibodies (or binding domains) or bispecific constructs described herein, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors. In another aspect, a nucleic acid molecule encoding any antibody (or binding domain) or bispecific construct described herein is provided. In another aspect, the nucleic acid molecule is in the form of an expression vector. In another aspect, the nucleic acid molecule is in the form of an expression vector that expresses the antibody (or binding domain) or bispecific construct when administered to a subject in vivo.

[0199] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region comprises an amino acid sequence set forth in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, or an amino acid sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the foregoing sequences). In another embodiment, the nucleic acid molecule comprises a nucleotide sequence set forth in Table 11, or a nucleotide sequence at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to one or more of the foregoing sequences).

[0200] In another embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy and light chain variable regions of the antibody, wherein the heavy and light chain variable regions comprise a combination of amino acid sequences set forth in Table 1B or Table 2B, or amino acid sequences at least 90% identical thereto (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto).

[0201] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian non-episomal vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0202] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in progeny due to either mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0203] G. Combination therapyAny antibody (or binding domain) and / or bispecific construct described herein can be administered in combination with an additional therapy, i.e., in combination with other agents. As used herein, the term "co-administered" includes any and all simultaneous, separate, or sequential administration of an antibody (or binding domain) and / or bispecific construct described herein with one or more additional therapeutic agents, including administration as part of a dosing regimen.For example, combination therapy can include combining any antibody (or binding domain) or bispecific construct described herein with an immunosuppressant (e.g., a corticosteroid, a nonsteroidal glucocorticoid receptor agonist, a leukotriene D4 antagonist, a leukotriene B4 antagonist, an A2A agonist, an A2B antagonist, a dopamine receptor agonist, pirfenidone, nintedanib, or an avB6 antagonist), Anti-IL-6, cyclophosphamide, methotrexate, mycophenolate mofetil, autologous hematopoietic stem cell transplant, B cell depleting agents (rituximab, inebilizumab, belimumab), abatacept, anti-TGFb, anti-IL33, anti-IL25, DMARDs (disease-modifying antirheumatic drugs), bronchodilators (e.g., beta-2 adrenergic receptor agonists, muscarinic antagonists, short-acting beta-2 receptor agonists, long-acting beta-2 receptor agonists, short-acting anticoagulants, anticholinergic drugs, methylxanthine drugs, long-acting anticholinergic drugs), other cytokine or cytokine receptor antagonists or antibodies (e.g., IL-13 antagonist, IL-6 antagonist, IL-1, IL-33, IL-25, or TNF-α antagonist, anti-IgE antibody, anti-IL31 antibody, anti-IL31R antibody, anti-IL13 antibody, anti-endoglin antibody, anti-IL1b antibody, another anti-TSLP antibody or anti-hTSLPR antibody, IL-5, anti-IgE , anti-IL4Ra), antibiotics, radiation therapy, leukotriene antagonists (e.g., montelukast, zafirlukast, or pranlukast), PDE4 inhibitors (e.g., roflumilast, xanthene), antihistamines, or antitussives; optionally, the antibody or antigen-binding fragment thereof is administered sequentially or simultaneously with the additional therapeutic agent.

[0204] Additional agents that delete or inhibit immunosuppressive activity, e.g., immunosuppressive activity by immune cells (e.g., regulatory T cells, NKT cells, macrophages, myeloid-derived suppressor cells, immature dendritic cells, or suppressive dendritic cells), that may be administered with the antibodies (or binding domains) or bispecific constructs described herein include, for example, antibodies and small molecule drugs, e.g., IDO inhibitors such as 1-methyltryptophan or derivatives, and immunosuppressants such as rapamycin, cyclosporine, and FK506; anti-TNF agents such as etanercept, adalimumab, and infliximab, and steroids. Examples of named natural and synthetic steroids include, for example, aldosterone, beclomethasone, betamethasone, budesonide, cloprednol, cortisone, cortivazol, deoxycortone, desonide, desoximetasone, dexamethasone, difluorocortolone, fluclorone, flumethasone, flunisolide, fluocinolone, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluorometholone, flurandrenolone, fluticasone, halcinonide, hydrocortisone, icomethasone, meprednisone, methylprednisolone, paramethasone, prednisolone, prednisone, tixocortol, and triamcinolone.

[0205] H. Uses and Methods of the Invention Provided herein are methods for treating diseases or conditions associated with the expression and / or activity of TSLP and / or SCF by administering to a patient in need thereof an antibody (or binding domain) or bispecific construct or composition described herein. For example, the methods provided herein are used to treat diseases or conditions associated with an immune response (e.g., immune cell migration, activation, and / or proliferation) via the interaction (e.g., binding) of TSLP and / or SCF with their receptors on immune cells (TSLPR and / or c-Kit, respectively).

[0206] The terms "treat," "treating," and "treatment," as used herein, refer to therapeutic or preventative measures described herein. Methods of "treatment" employ administering an antibody (or binding domain), bispecific construct, or composition described herein to a subject in need of such treatment, e.g., a subject in need of an enhanced immune response to a particular antigen or a subject who may ultimately acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or alleviate one or more symptoms of the disorder or recurring disorder, or to prolong the survival of the subject beyond that which would be expected in the absence of such treatment.

[0207] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The amount effective for this use will depend on the severity of the disorder being treated and the general status of the patient's own immune system.

[0208] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0209] As used herein, the term "immune cells" includes cells that are of hematopoietic origin and play a role in the immune response, e.g., lymphocytes such as B cells and T cells; natural killer cells; myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0210] As used herein, the term "immune response" refers to the action of immune cells (such as lymphocytes, antigen-presenting cells, phagocytes, or granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by immune cells or the liver, which result in the selective damage, destruction, or removal from the human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or, in cases of autoimmune or pathological inflammation, normal human cells or tissues. As used herein, the term "antigen-specific T cell response" refers to an immune response generated by a T cell when the T cell is stimulated by an antigen specific for the T cell. Non-limiting examples of responses generated by T cells in response to antigen-specific stimulation include T cell proliferation and cytokine (e.g., IL-2) production.

[0211] Thus, in some aspects, the disclosure provides methods for blocking the binding of TSLP and / or SCF to their receptors (i.e., TLSPR and c-Kit, respectively) in a subject, comprising administering to the subject any one of the antibodies (or binding domains), bispecific constructs, or compositions described herein in an amount effective to block the binding of TSLP and / or SCF to their receptors.

[0212] As used herein, the terms "blocking the binding of TSLP and / or SCF" and "inhibiting the binding of TSLP and / or SCF" (e.g., with respect to the binding of TSLP to TSLPR and / or the binding of SCF to c-Kit) are used interchangeably and are intended to include any measurable reduction in the binding between a ligand (TSLP or SCF) and its receptor (TSLPR or c-Kit), e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% blocking of the binding of TSLP and / or SCF to TSLPR and / or c-Kit.

[0213] Also provided herein are methods for inhibiting immune cell activation in a subject, and for reducing or preventing immune cell accumulation in an organ or tissue in a subject, comprising administering to the subject any one of the antibodies (or binding domains), bispecific constructs, or compositions described herein in an amount effective to inhibit immune cell activation in the subject or reduce immune cell accumulation in an organ or tissue in the subject.

[0214] As used herein, the terms "inhibiting immune cell activation" and "reducing immune cell accumulation" (e.g., with respect to cells associated with immune function such as cytokines) are used interchangeably and are intended to include any measurable decrease in the amount of immune cells and / or signaling between immune cells within a tissue or organ, e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% reduction in immune cell activation and / or accumulation. In one aspect, reduced immune cell activation and / or accumulation results in inhibition or blocking of (a) TSLP-induced activation and / or proliferation of mast cells, DCs, and / or NKT cells, (b) TSLP-induced osteoprotegerin (OPG) secretion, (c) TSLP-induced secretion of Th2 cytokines (such as TARC, CCL22, IL-4, IL-13, or IL-5), and / or (d) SCF-induced secretion of mast cells, eosinophils, type 2 innate lymphoid cells (ILC2) cells, and / or type 3 innate lymphoid cells (ILC3) cells. Overall, by inhibiting or preventing the activation of immune cells and reducing or preventing their accumulation in organs or tissues, various diseases and disorders involving inflammation are treated or prevented, such as autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, pulmonary diseases, metabolic diseases (such as type 2 diabetes), neurodegenerative diseases (such as Parkinson's disease), certain types of cancer (such as colon cancer), and psychiatric illnesses (such as depression), as well as allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 responses, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, and food allergies, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorders, and rhinoconjunctivitis. Other conditions and disorders include fibrotic diseases and illnesses associated with tissue remodeling, such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, liver fibrosis, fibromyalgia, gingival fibrosis, or radiation-induced fibrosis.

[0215] Also provided herein are methods of reducing or inhibiting inflammation in a subject in need thereof, as well as methods of treating inflammatory diseases or disorders (e.g., autoimmune diseases, cardiovascular diseases, gastrointestinal diseases, pulmonary diseases, metabolic diseases (such as type 2 diabetes), neurodegenerative diseases (such as Parkinson's disease), certain types of cancer (such as colon cancer), and psychiatric diseases (such as depression)) by administering to a patient in need thereof a construct, antibody or antigen-binding fragment thereof, bispecific construct, or composition described herein in an amount effective to inhibit or reduce inflammation in the subject.

[0216] As used herein, the terms "inhibiting inflammation" and "reducing inflammation" (e.g., with respect to bodily tissue, i.e., muscle tissue, epithelial tissue, connective tissue, and nervous tissue) are used interchangeably and are intended to include any measurable decrease in the response of bodily tissue involving immune cells, blood vessels, and molecular mediators to harmful stimuli (such as pathogens, damaged cells, or irritants), e.g., at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% reduction in inflammation in the bodily tissue.

[0217] In one aspect, a method for reducing inflammation is provided, the method comprising contacting a body tissue with any one of the antibodies (or binding domains), bispecific constructs, or compositions described herein. Reducing inflammation can include, for example, inhibiting the binding of human TSLP to the human TSLP receptor (TSLP-R), inhibiting the binding of SCF-binding to c-Kit, inhibiting the proliferation of BaF3 cells, inhibiting TARC induction in human dendritic cells, inhibiting the phosphorylation of c-Kit in human CHO-KIT cells, inhibiting the degranulation of human mast cells, and / or inhibiting the proliferation of M-07e cells.

[0218] In another aspect, a method is provided for treating a condition or disease in a subject, the method comprising administering to the subject any one of the antibodies (or binding domains), bispecific constructs, or compositions described herein in an amount effective to treat the condition or disease.

[0219] The subject may be, for example, a subject suffering from a condition or disease that is desired to reduce inflammation.In one embodiment, the condition or disease is related to the migration, activation, and / or proliferation of immune cells through the interaction (e.g., binding) of TSLP and / or SCF with the receptor on immune cells (TSLPR and / or c-Kit, respectively), such as immune system disorders, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, and food allergy, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorder, and rhinoconjunctivitis. Other conditions and disorders include fibrotic diseases and illnesses associated with tissue remodeling, such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, liver fibrosis, fibromyalgia, gingival fibrosis, or radiation-induced fibrosis.

[0220] In another aspect, a method for treating a condition or disease in a subject is provided, wherein the method comprises administering to the subject any one of the anti-TSLP antibodies or antigen-binding fragments thereof described herein in combination with any one of the anti-SCF antibodies or antigen-binding fragments thereof described herein. For example, to treat a condition or disease in a subject, any one of the anti-TSLP antibodies or antigen-binding fragments thereof according to those listed in Table 1A or 1B can be combined with any one of the anti-SCF antibodies or antigen-binding fragments thereof according to those listed in Table 2A or 2B.

[0221] In one aspect, an antibody (or binding domain), bispecific construct, or composition described herein is administered in combination with an immunosuppressant (e.g., a corticosteroid, a nonsteroidal glucocorticoid receptor agonist, a leukotriene D4 antagonist, a leukotriene B4 antagonist, an A2A agonist, an A2B antagonist, a dopamine receptor agonist, pirfenidone, nintedanib, or an avB6 antagonist), a bronchodilator (e.g., a beta-2 adrenergic receptor agonist, a muscarinic antagonist, a short-acting beta-2 receptor agonist, a long-acting beta-2 receptor agonist, a short-acting anticholinergic, a methylxanthine drug, a long-acting anticholinergic), or other anti-inflammatory drug. is administered in combination with one or more additional therapeutic agents selected from, but not limited to, a kine or cytokine receptor antagonist or antibody (e.g., an IL-13 antagonist, an IL-6 antagonist, an IL-1, IL-33, IL-25, or TNF-α antagonist, an anti-IgE antibody, an anti-IL31 antibody, an anti-IL31R antibody, an anti-IL13 antibody, an anti-endoglin antibody, an anti-IL1b antibody, another anti-TSLP antibody, or an anti-hTSLPR antibody), an antibiotic, radiation therapy, a leukotriene antagonist (e.g., montelukast, zafirlukast, or pranlukast), a PDE4 inhibitor (e.g., roflumilast, xanthene), an antihistamine, or an antitussive; Optionally, the antibody (or binding domain), bispecific construct or composition is administered sequentially or simultaneously with an additional therapeutic agent.

[0222] I. kit Also provided are kits (e.g., diagnostic kits) comprising one or more anti-TSLP antibodies (or binding domains), anti-SCF antibodies (or binding domains), bispecific constructs, or compositions described herein, optionally together with instructions for use. The kits may also include an informational brochure, e.g., a brochure informing users how to use the reagents to practice the methods disclosed herein. The term "brochure" includes any written, marketing, or recorded material supplied on or with the kit, or otherwise accompanying the kit.

[0223] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. [Example]

[0224] V. Working Examples Example 1: Generation of TSLP-specific monoclonal antibodies immunization : Mouse anti-TSLP monoclonal antibodies were generated by immunizing BALB / c mice with soluble human TSLP antigen.

[0225] Antigen and immunization: The antigen (a soluble fusion protein containing a HIS-tagged TSLP protein (AcroBiosystems®)) was mixed with the MPL plus TDM adjuvant system (Sigma®). Five to 20 micrograms of soluble recombinant TSLP antigen in PBS was mixed 1:1 with the adjuvant. Two hundred microliters of the prepared antigen was injected into the peritoneal cavity of mice approximately every 14 to 30 days. Animals that developed anti-TSLP titers were given an iv injection of 1 to 10 micrograms of soluble recombinant TSLP antigen 3 to 4 days before fusion. Mouse spleens were harvested, and isolated splenocytes were used to prepare hybridomas.

[0226] fusion The P3x63Ag8.653 mouse myeloma cell line (ATCC® CRL 1580) was used for fusion. RPMI 1640 (Invitrogen®) containing 10% FBS was used for culturing the myeloma cells. Hybridoma growth medium was supplemented with additional media supplements, including: HAT (Sigma; 1.0 × 10 4 M Hypoxanthine, 4.0 × 10 -7 M aminopterin, 1.6 × 10 -5 M-Thymidine medium containing up to 10% Hybridoma Cloning Supplement (Sigma®), 10% FBS (Sigma), L-glutamine (Gibco®), 0.1% gentamicin (Gibco), 2-mercaptoethanol (Gibco).

[0227] Splenocytes were mixed with P3x63Ag8.653 myeloma cells at a 6:1 ratio and pelleted by centrifugation. To promote fusion, polyethylene glycol was added dropwise with careful mixing. Hybridomas were cultured for 2–3 weeks until visible colonies were established. Supernatants were harvested and used for initial screening of mouse IgG via ELISA using a human soluble TSLP fusion protein and mouse Fc-specific detection. IgG-positive supernatants were then assayed for TSLP blockade by ELISA. Hybridomas were also screened for cross-reactivity with cynomolgus macaque TSLP.

[0228] Hybridoma cells were expanded and cell pellets were frozen for RNA isolation and sequencing. H Coding region and V LThe coding region was identified using RNA from the corresponding hybridoma. The RNA was reverse transcribed into cDNA, and the V coding region was amplified by PCR. The PCR product was sequenced, inserted into a human IgG1 vector, transiently expressed as a chimeric antibody, and purified by Protein A column chromatography. Antibody 1D10 was isolated.

[0229] Example 2: Generation of humanized TSLP antibodies Computer models of the parent heavy and light chain variable region domains (i.e., VH and VL domains) of antibody 1D10 from Example 1 were generated and used to guide the humanization process. The parent VH and VL sequences were aligned with a panel of human germline sequences that had been filtered to select germline sequences that did not contain undesirable sequence liabilities, particularly N-linked glycosylation sites and free cysteines. The best germline matches were selected from two different VH and VL families. A humanization algorithm was then used to select CDR and framework amino acids for grafting from the donor parent sequence onto the human acceptor germline sequence (Table 4). The complementarity-determining regions (CDRs) from the parent antibody were grafted onto the appropriate human framework, and back mutations were introduced (Tables 4A and 4B; underlined amino acids differ from the parent amino acids).

[0230] (Table 4) Clone 1D10 TIFF2026508389000007.tif65150

[0231] (Table 5A) VH CDR sequences TIFF2026508389000008.tif37156TIFF2026508389000009.tif215156

[0232] Table 5B: VL CDR sequences TIFF2026508389000010.tif215156TIFF2026508389000011.tif28156

[0233] Four heavy chain and four light chain humanized variants were designed for antibody 1D10. The 1D10 heavy chain variants were designated 1D10-H1 (SEQ ID NO: 1), 1D10-H2 (SEQ ID NO: 5), 1D10-H3 (SEQ ID NO: 9), and 1D10-H4 (SEQ ID NO: 13). The 1D10 light chain variants were designated 1D10-L1 (SEQ ID NO: 17), 1D10-L2 (SEQ ID NO: 21), 1D10-L3 (SEQ ID NO: 25), and 1D10-L4 (SEQ ID NO: 29). The pairing of these variable domain sequences is shown in Table 4. The activities of these antibodies and sequences were further investigated as described below.

[0234] Table 6: Heavy and light chain pairing TIFF2026508389000012.tif69146

[0235] Example 3: Binding to human TSLP Microtiter plates were coated with recombinant human TSLP-kappa (Celldex Therapeutics, Inc.®) in PBS and then blocked with 5% bovine serum albumin in PBS. Protein A-purified chimeric mAb 1D10, its humanized version, and an isotype control were added at various concentrations and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative binding curves are shown in Figures 1-3.

[0236] Example 4: Blocking TSLP binding to TSLP-R The ability of anti-TSLP human mAbs to block the binding of human TSLP to the human TSLP receptor (TSLP-R) was investigated by ELISA as follows.

[0237] Microtiter plates were coated with recombinant human TSLP-R (AcroBiosystems) in PBS and then blocked with 5% bovine serum albumin in PBS. Various concentrations of protein A-purified chimeric mAb 1D10, its humanized version, and an isotype control were preincubated with biotinylated recombinant human TSLP (AcroBiosystems) for 20 minutes at room temperature and then added to the plates and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with strepavidin conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative curves are shown in Figures 4-6, demonstrating the ability of anti-TSLP human mAbs to block the binding of human TSLP to the human TSLP receptor (TSLP-R).

[0238] Example 5: Inhibition of proliferation of BaF3 cells BaF3 cells were transfected to express human TSLP and human IL-7Ra on their surface. Cells were incubated with recombinant human TSLP (R&D Systems®) in the presence of medium, Protein A-purified chimeric mAb 1D10, selected humanized versions, or isotype controls at 37°C and 6% CO2. After 3 days, CellTiter Glo (Promega®) was added according to the kit's instructions, and luminescence resulting from cell proliferation was detected and quantified using a Perkin Elmer Victor X4 luminometer. Representative curves are shown in Figure 7, demonstrating the ability of anti-TSLP human mAbs to inhibit BaF3 proliferation.

[0239] Example 6: Inhibition of TARC induction in human dendritic cells Human dendritic cells (DCs) were isolated from pre-frozen peripheral blood mononuclear cell leukopacks (BioIVT®) using the MACS Cell Separation Pan DC Enrichment Kit from Miltenyi Biotec®. Cells were incubated overnight at 37°C and 6% CO2 with recombinant human TSLP (R&D Systems®) in the presence of medium, protein A-purified chimeric mAb 1D10, selected humanized versions, or an isotype control. Supernatants were harvested, and TARC production was quantified by ELISA (R&D Systems). Representative curves are shown in Figure 8, demonstrating the ability of anti-TSLP human mAbs to inhibit TARC induction in human DCs.

[0240] Example 7: Affinity and kinetic constants of humanized mAbs The binding affinity and binding kinetics of various human anti-TSLP antibodies were determined using Octet® QK according to the manufacturer's guidelines. e The samples were examined by Bio-Layer Interferometry (BLI®) using an instrument (Sartorius®).

[0241] To assess affinity for human TSLP, purified antibodies were captured on an Anti-Human Fc Capture (AHC) biosensor (Sartorius). The sensor was preconditioned by two cycles of association and regeneration using an irrelevant HuIgG1 antibody. Each anti-TSLP antibody was adjusted to 0.5–1.0 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and loaded onto a freshly hydrated and preconditioned AHC biosensor for 300 seconds at 30°C and a plate shaking speed of 1000 rpm. Eight biosensors were loaded with the same antibody per assay.

[0242] Binding was determined by exposing seven antibody-loaded biosensors to either soluble human TSLP-HIS or soluble cynomolgus monkey TSLP (AcroBiosciences). Affinity measurements were performed at 30°C and 1000 rpm using two-fold serial dilutions of the analyte ranging from 25 to 0.4 nM in dilution buffer. Association of the antibody-loaded biosensors in the analyte wells was performed for 300 seconds, and then the biosensors were transferred to the dilution buffer wells for 900 seconds for dissociation measurements. Both association and dissociation steps were performed at 30°C and 1000 rpm.

[0243] For cynomolgus TSLP experiments, soluble cynomolgus TSLP-HIS (ACROBiosystems) was prepared at 2.0 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and loaded onto freshly hydrated and regenerated Anti-Penta-HIS (HIS1K) biosensors (Sartorius) for 300 seconds at 30°C and a plate shaking speed of 1000 rpm. Eight biosensors were loaded with the same ligand per assay.

[0244] Cynomolgus TSLP binding was determined by exposing seven ligand-loaded biosensors to the analyte, a purified anti-TSLP antibody. Affinity measurements were obtained using two-fold serial dilutions of the analyte ranging from 50 to 3.1 nM in dilution buffer. Association of the ligand-loaded biosensors in the analyte wells was performed for 180 seconds, and then the biosensors were transferred to the dilution buffer wells for 300 seconds for dissociation measurements. Both association and dissociation steps were performed at 30°C and a plate shaking speed of 1000 rpm.

[0245] A corresponding control was performed in each case by keeping one biosensor capturing the ligand in the dilution buffer well during the association and dissociation steps. The control biosensor data were used to subtract the background and to account for biosensor drift and dissociation of the ligand from the biosensor.

[0246] Octet BLI Analysis Software (Sartorius) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer that bound to the captured ligand. Association and dissociation curves were fitted to a 1:1 binding model using the data analysis software according to the manufacturer's guidelines.

[0247] The determined affinity and kinetic parameters (background subtracted) for human TSLP and cynomolgus TSLP are shown in Figures 9A and 9B, where k on = association velocity, k dis = dissociation rate, and KD = ratio k dis / k on is the affinity constant determined by

[0248] Example 8: Generation of SCF-specific monoclonal antibodies Mice were immunized with human SCF protein and boosted with the protein. Immune responses were tested by ELISA against human SCF protein. Plasma cells from mice with high antibody titers were isolated using a CD138 B cell enrichment kit, loaded onto a Beacon 14K chip, and cloned using a Berkeley Lights® Beacon instrument. Bead-based screening on the Beacon chip was performed on human, cynomolgus monkey, and mouse SCF-specific IgG-secreting B cells to identify single B cells with blocking activity against the ECD of the human KIT receptor. Single B cells were selected and transferred to lysis buffer for single-cell sequencing in 96-well plates. This was performed by RNA purification, reverse transcription, and cDNA amplification, VH and VL amplification, and VH and VL sequencing and analysis. A human IgG1 chimeric antibody, mAb12 (SCF-12), was selected and purified after expression.

[0249] Example 9: Generation of humanized SCF antibodies Computer models of the parent heavy and light chain variable region domains (i.e., VH and VL domains) of antibody mAb12 from Example 8 were generated and used to guide the humanization process. The parent VH and VL sequences were aligned with a panel of human germline sequences that had been filtered to select germline sequences that did not contain undesirable sequence liabilities, particularly N-linked glycosylation sites and free cysteines. The best germline matches were selected from two different VH and VL families. A humanization algorithm was then used to select CDR and framework amino acids for grafting from the donor parent sequence onto the human acceptor germline sequence (Table 7). The complementarity-determining regions (CDRs) from the parent antibody were grafted onto the appropriate human framework, with back mutations introduced where necessary (Tables 7A and 7B; underlined amino acids differ from the parent amino acids).

[0250] Table 7 Clone mAb12 TIFF2026508389000013.tif81156

[0251] Table 8A: VH CDR sequences TIFF2026508389000014.tif73156TIFF2026508389000015.tif223156TIFF2026508389000016.tif82156

[0252] Table 8B: VL CDR sequences TIFF2026508389000017.tif129156TIFF2026508389000018.tif129156

[0253] Four heavy chain and four light chain humanized variants were designed for the antibody mAb12. The mAb12 heavy chain variants were designated mAb12-H1 (SEQ ID NO: 33), mAb12-H2 (SEQ ID NO: 37), mAb12-H3 (SEQ ID NO: 41), and mAb12-H4 (SEQ ID NO: 45). The mAb12 light chain variants were designated mAb12-L1 (SEQ ID NO: 49), mAb12-L2 (SEQ ID NO: 53), mAb12-L3 (SEQ ID NO: 57), and mAb12-L4 (SEQ ID NO: 61). The pairing of these variable domain sequences is shown in Table 9. The activities of these antibodies and sequences were further investigated as described below.

[0254] Table 9: Heavy and light chain pairings TIFF2026508389000019.tif62157

[0255] Example 10: Binding to human SCF Microtiter plates were coated with recombinant human SCF-HIS (Celldex) in PBS and then blocked with 5% bovine serum albumin in PBS. Protein A-purified chimeric mAb designated mAb12, its humanized version, and an isotype control were added at various concentrations and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative binding curves are shown in Figures 10 and 11.

[0256] Example 11: Blocking the binding of SCF to c-Kit The ability of anti-SCF human mAbs to block the binding of human SCF to human c-Kit was investigated by ELISA as follows.

[0257] Microtiter plates were coated with recombinant human c-Kit-Fc (Celldex) in PBS and then blocked with 5% bovine serum albumin in PBS. Various concentrations of protein A-purified chimeric mAb12, its humanized version, and an isotype control were preincubated with biotinylated recombinant human SCF (AcroBiosystems) at room temperature for 20 minutes, then added to the plate and incubated at 37°C. The plate was washed with PBS / Tween and then incubated with strepavidin conjugated to horseradish peroxidase at 37°C. After washing, the plate was developed with HRP substrate and analyzed at OD 450nm using a microtiter plate reader. Representative curves are shown in Figures 12 and 13, which show the ability of anti-SCF human mAbs to block the binding of SCF to its receptor c-KIT.

[0258] Example 12: Affinity and kinetic constants of humanized mAbs The binding affinity and binding kinetics of the human anti-SCF antibodies were determined using Octet® QK according to the manufacturer's guidelines. e The samples were examined by biolayer interferometry (BLI) using a Sartorius instrument.

[0259] Purified antibodies were captured on an Anti-Human Fc Capture (AHC) biosensor (Sartorius). The sensor was preconditioned by two cycles of association and regeneration using an irrelevant HuIgG1 antibody. Each anti-SCF antibody was prepared at 0.5 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and loaded onto a freshly hydrated and preconditioned AHC biosensor for 300 seconds at 30°C and a plate shaking speed of 1000 rpm to achieve a target response of 0.7 nm. Eight biosensors were loaded with the same antibody per assay.

[0260] Binding was determined by exposing seven antibody-loaded biosensors to soluble human SCF-HIS or soluble cynomolgus SCF analytes. Affinity measurements were performed at 30°C and 1000 rpm using two-fold serial dilutions of analyte ranging from 800 to 6.25 nM in dilution buffer. Association of the antibody-loaded biosensors in the analyte wells was performed for 300 seconds, and then the biosensors were transferred to the dilution buffer wells for 900 seconds for dissociation measurements.

[0261] A corresponding control was performed in each case by keeping the remaining biosensor capturing the antibody in the dilution buffer well during the association and dissociation steps. The control biosensor data were used to subtract background and to account for biosensor drift and dissociation of the antibody from the biosensor.

[0262] The Octet BLI Analysis Software system (Sartorius) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer that bound to the captured antibody. Association and dissociation curves were fitted to a 1:1 binding model using the data analysis software according to the manufacturer's guidelines.

[0263] The affinity and kinetic parameters (background subtracted) for human SCF and cynomolgus SCF are shown in Figures 14A and 14B, where k on = association velocity, k dis = dissociation rate, and K D = ratio k dis / k on is the affinity constant determined by

[0264] Example 13: Inhibition of c-Kit phosphorylation in human CHO-KIT cells Standard MSD plates were coated with purified anti-human CD117 (c-Kit) antibody. The plates were sealed and shaken at 500 rpm for 10 minutes, then incubated overnight at 4°C. 100,000 CHO cells overexpressing human c-Kit were seeded into 96-well tissue culture-treated plates at 100,000 cells / well and incubated overnight at 37°C / 5% CO2. Diluted anti-SCF antibody was added to the appropriate wells and incubated for 2 hours at 37°C / 5% CO2. The antibody coating was removed from the MSD plates, washed, and then blocked with TBST / 5% BSA for 1 hour at room temperature with shaking at 500 RPM. Diluted recombinant human SCF was added to all wells and then incubated for 10 minutes at 37°C / 5% CO2. After incubation, cells were lysed in ice-cold PBS / 0.1% Triton X-100 supplemented with phosphatase and protease inhibitors and shaken at 4°C for 5 minutes. The blocked MSD plate was washed with TBST, and the cell lysate was applied to the MSD plate and shaken at 500 rpm for 1 hour at room temperature. SulfoTag-pY20 detection antibody was added to all wells and shaken at 500 rpm for 1 hour at room temperature. Results were read in 1x Read Buffer using an MSD Sector Plate Reader. A representative curve is shown in Figure 15, which shows the ability of anti-SCF human mAb to inhibit c-KIT phosphorylation in human CHO-KIT cells.

[0265] Example 14: Inhibition of human mast cell degranulation Mature human mast cells were cytokine-starved overnight at 37°C / 5% CO2. The next day, the mast cells were washed, resuspended in warm HEPES buffer, and transferred to a 96-well tissue culture plate. Anti-SCF antibodies were diluted and incubated with human mast cells for 1 hour at 37°C in air. Human IgE was then added to the human mast cell / antibody mixture for 30 minutes at 37°C in air. After IgE treatment, human SCF and goat anti-human IgE were diluted, added to the appropriate wells to induce cross-linking, and incubated with human mast cells for 30 minutes at 37°C in air. After incubation, the samples were diluted with PNAG solution and incubated for 90 minutes at 37°C in air. β-hexosaminidase was then read by adding glycine buffer and analyzed at OD 405 nm using a microtiter plate reader. A representative curve is shown in Figure 16, demonstrating the ability of anti-SCF human mAbs to inhibit human mast cell degranulation.

[0266] Example 15: Inhibition of proliferation of M-07e cells M-07e cells were incubated with dilutions of anti-SCF antibodies or isotype controls at 37°C and 5% CO2 for 1 hour. After incubation, recombinant human SCF (R&D Systems) was added to all wells and incubated at 37°C and 6% CO2 for 6 days. After 6 days, CellTiter Glo (Promega) was added according to the kit's instructions, and luminescence resulting from cell proliferation was detected and quantified using a microtiter plate reader. A representative curve is shown in Figure 17, which shows the ability of anti-SCF human mAb to inhibit M-07e cell proliferation.

[0267] Example 16: Epitope Mapping Microtiter plates were coated with recombinant human wild-type SCF-HIS (Celldex) or mutant versions of SCF in PBS and then blocked with 5% bovine serum albumin in PBS. Protein A-purified humanized mAb12 was added at various concentrations and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative binding curves are shown in Figures 18A and 18B. A three-dimensional drawing showing SCF binding to KIT-D1, including residue K100, is provided in Figure 18C.

[0268] Example 17: S1 / S14 SCF cell binding SCF 220 Murine S1 / S14 cells (ATCC), engineered to overexpress a membrane-bound form of SCF known as SCF, were incubated with diluted humanized mAb12 or human KIT-ECD for 1 hour at 4°C. The cells were washed and incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to an R-phycoerythrin fluorophore for 1 hour at 4°C. After incubation, the cells were washed and fixed. Mean fluorescence intensity was read using a Bectin Dickonson® (BD) Accuri C6 Plus Personal Flow Cytometer instrument.

[0269] Murine S1 / S14 cells (ATCC), engineered to overexpress the soluble SCF isoform known as SCF-248, were incubated with diluted humanized mAb12 or human KIT-ECD for 1 hour at 4°C. The cells were washed and incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to an R-phycoerythrin fluorophore for 1 hour at 4°C. After incubation, the cells were washed and fixed. Mean fluorescence intensity was read using a BD Accuri C6 Plus Personal Flow Cytometer.

[0270] Representative binding curves are shown in Figures 19A and 19B, which demonstrate that humanized mAb12 preferentially binds to and blocks soluble SCF.

[0271] Example 18: Inhibition of c-Kit phosphorylation in human M-07e cells Soluble and SCF 220 Inducible phosphorylation was assessed by ELISA. 220 Cells were treated with mitomycin C for 2 hours, washed, and allowed to recover overnight at 37°C and 5% CO2. M-07e cells were washed and serum-starved overnight at 37°C and 5% CO2. Diluted mAb12 was added to soluble SCF and parental S1 / S14 cells, or S1 / S14-SCF, the source of SCF used to stimulate M-07e cells. 220The cells were preincubated with either S1 / S14 cells or S1 / S15 cells at 37°C and 5% CO2 for 1 hour. After preincubation, M-07e cells were added to S1 / S14 cells and spun down at 1200 rpm for 5 minutes, followed by incubation at 37°C and 5% CO2 for 20 minutes. After incubation, the medium was removed, and the cells were lysed in 2x lysis buffer at 4°C for 10 minutes. Recombinant human c-Kit was used to coat a 96-well high-binding plate, followed by blocking with 5% BSA. The cell lysate was added to the c-Kit-coated plate and incubated at room temperature. The plate was washed with PBS / Tween and then incubated with HRP-conjugated pY20 detection antibody. After washing, the plate was developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. A representative curve is shown in Figure 20, which shows the ability of anti-SCF human mAb to inhibit c-KIT phosphorylation in human M-07e cells.

[0272] Example 19: Construction and production of bispecific antibodies (CDX-622) A tetravalent bispecific antibody construct was developed using a mutated fully human IgG1 backbone for the TSLP monoclonal antibody sequences (1D10 VH1-L1 and 1D10 VH2-L1) and the scFv of the SCF monoclonal antibody genetically linked to the C-terminus of the 1D10 heavy chain via a linker (Figures 21A and 21B). The humanized antibody scFv sequences used in the bispecific were taken from mAb12 VH1-L4, mAb12 VH2-L4, and mAb12 VH4-L3. The Fc domain was mutated (234A, 235Q, 322Q, 252Y, 254T, and 256E).

[0273] The constant domain sequence was as follows (mutations shown in bold): TIFF2026508389000020.tif44145

[0274] The bispecific constructs were expressed in CHO cell lines. Table 10 defines the constructs.

[0275] Table 10. Pairing of IgG and scFv The TIFF2026508389000021.tif451405.7 construct ((1D10(VH2-L1)-mAb12(VH4-L3)) was designated "CDX-622" and further analyzed as described in the next example.

[0276] Example 20: Binding to human TSLP Microtiter plates were coated with recombinant human TSLP-kappa (Celldex) in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of bispecific antibodies and isotype controls were added and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative curves are shown in Figure 22, which demonstrate the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to bind to human TSLP.

[0277] Example 21: Binding to human SCF Microtiter plates were coated with recombinant human SCF-HIS (Celldex) in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of bispecific antibodies and isotype controls were added and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG Fc-specific polyclonal reagent conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative curves are shown in Figure 23, which demonstrate the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to bind to human SCF.

[0278] Example 22: Bifunctional binding to human TSLP and human SCF Microtiter plates were coated with recombinant human TSLP-kappa in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of the bispecific antibody and isotype control were allowed to bind to TSLP, followed by the addition of biotinylated human SCF (AcroBiosystems), which was detected with HRP-labeled strepavidin. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. A representative curve is shown in Figure 24, demonstrating the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to simultaneously bind to human TSLP and human SCF.

[0279] Example 23: Blocking TSLP binding to TSLP-R The ability of the bispecific antibodies to block the binding of human TSLP to the human TSLP receptor (TSLP-R) was investigated by ELISA as follows.

[0280] Microtiter plates were coated with recombinant human TSLP-R (AcroBiosystems) in PBS and then blocked with 5% bovine serum albumin in PBS. Dilutions of bispecific antibodies and isotype controls were preincubated with biotinylated recombinant human TSLP (AcroBiosystems) for 20 minutes at room temperature, then added to the plates and incubated at 37°C. The plates were washed with PBS / Tween and then incubated with strepavidin conjugated to horseradish peroxidase at 37°C. After washing, the plates were developed with HRP substrate and analyzed at OD 450 nm using a microtiter plate reader. Representative curves are shown in Figures 25A and 25B, which demonstrate the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to block the binding of TSLP to its receptor, TSLP-R.

[0281] Example 24: Blocking the binding of SCF to c-Kit The ability of anti-SCF human mAb to block the binding of human SCF expressed on the surface of MO7e cells to human c-Kit was investigated as follows.

[0282] Dilutions of bispecific antibodies and dilutions of isotype controls are pre-incubated with biotinylated recombinant human SCF (AcroBiosystems) at room temperature for 20 minutes, then added to cells and incubated on a plate shaker at room temperature.Cells are washed and incubated with strepavidin conjugated to phycoerythrin at room temperature on a plate shaker.After washing, cell-associated fluorescence is determined by analysis using a FACSCanto II™ instrument (BD Biosciences) according to the manufacturer's instructions.Representative curves are shown in Figure 26, which show the ability of bispecific constructs (i.e., the pairing of IgG and scFv shown in Table 10) to block the binding of SCF to its receptor c-KIT.

[0283] Example 25: Inhibition of proliferation of BaF3 cells BaF3 cells were transfected to express human TSLP and human IL-7Ra on their surface. In the presence of medium, dilutions of bispecific antibodies, or dilutions of isotype controls, cells were incubated with recombinant human TSLP (R&D Systems) at 37°C and 6% CO2. After 3 days, CellTiter Glo (Promega) was added according to the kit's instructions, and the luminescence resulting from cell proliferation was detected and quantified using a Perkin Elmer Victor X4® luminometer. Representative curves are shown in Figures 27A and 27B, which show the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to inhibit the proliferation of BaF3 cells.

[0284] Example 26: Inhibition of TARC induction in human dendritic cells Human dendritic cells (DCs) were isolated from pre-frozen peripheral blood mononuclear cell leukoplak (BioIVT) using the MACS Cell Separation Pan DC Enrichment Kit from Miltenyi Biotec®. Cells were incubated overnight with recombinant human TSLP (R&D Systems) in the presence of medium, diluent of bispecific antibody, or diluent of isotype control at 37°C and 6% CO2. Supernatants were collected, and thymus- and activation-regulated chemokine (TARC) production was quantified by ELISA (R&D Systems). Representative curves are shown in Figures 28A and 28B, which show the ability of Construct 5.2 and Construct 5.7 (CDX-622) to inhibit TARC release.

[0285] Example 27: Affinity and kinetic constants of humanized BsAbs The binding affinity and binding kinetics of the purified bispecific antibodies were assayed using Octet® QK according to the manufacturer's guidelines. e The samples were examined by biolayer interferometry (BLI) using a Sartorius instrument.

[0286] To assess the affinity for human TSLP, purified bispecific antibodies were captured on an Anti-Human Fc Capture (AHC) (Sartorius). Each bispecific antibody was adjusted to 1.0 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and loaded onto freshly hydrated biosensors for 300 seconds at 30°C and a plate shaking speed of 1000 rpm. Eight biosensors were loaded with the same antibody per assay.

[0287] Human TSLP binding was determined by exposing seven antibody-loaded biosensors to the analyte, soluble human TSLP-HIS (ACROBiosystems). Affinity measurements were obtained using two-fold serial dilutions of the analyte ranging from 12.5 to 0.8 nM in dilution buffer. Association of the antibody-loaded biosensors in the analyte wells was performed for 300 seconds, and then the biosensors were transferred to the dilution buffer wells for 1200 seconds for dissociation measurements. Both association and dissociation steps were performed at 30°C and a plate shaking speed of 1000 rpm.

[0288] For cynomolgus TSLP experiments, soluble cynomolgus TSLP-HIS (ACROBiosystems) was prepared at 2.0 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and loaded onto freshly hydrated Anti-Penta-HIS (HIS1K) biosensors (Sartorius) for 300 seconds at 30°C and a plate shaking speed of 1000 rpm. Eight biosensors were loaded with the same antigen per assay.

[0289] Cynomolgus TSLP binding was determined by exposing seven ligand-loaded biosensors to the analyte, a purified bispecific antibody. Affinity measurements were obtained using two-fold serial dilutions of the analyte ranging from 200 to 3.1 nM in dilution buffer. Association of the ligand-loaded biosensors in the analyte wells was performed for 180 seconds, and then the biosensors were transferred to the dilution buffer wells for 300 seconds for dissociation measurements. Both association and dissociation steps were performed at 30°C and a plate shaking speed of 1000 rpm.

[0290] To assess the affinity of purified bispecific antibodies to human SCF and cynomolgus monkey SCF, the antibodies were captured on an Anti-Human Fab-CH1 2nd Generation (FAB2G) biosensor (Sartorius). Each bispecific antibody was prepared at 1.0 μg / mL in dilution buffer (10 mM PO4 + 150 mM NaCl + 1 mg / mL BSA + 0.05% Tween 20, pH 7.2) and loaded onto a freshly hydrated biosensor for 300 seconds at 30°C and a plate shaking speed of 1000 rpm. Eight biosensors were loaded with the same antibody per assay.

[0291] The binding of human SCF and cynomolgus SCF was determined by exposing seven antibody-loaded biosensors to soluble human SCF-HIS or soluble cynomolgus SCF-HIS analytes. Affinity measurements were obtained using two-fold serial dilutions of the analyte ranging from 800 to 12.5 nM in dilution buffer. Association of the antibody-loaded biosensors in the analyte wells was performed for 300 seconds, followed by transfer of the biosensors to the dilution buffer wells for 900 to 1200 seconds for dissociation measurements. Both association and dissociation steps were performed at 30°C and a plate shaking speed of 1000 rpm.

[0292] A corresponding control was performed in each case by keeping one biosensor capturing the ligand in the dilution buffer well during the association and dissociation steps. The control biosensor data were used to subtract the background and to account for biosensor drift and dissociation of the ligand from the biosensor.

[0293] Octet BLI Analysis Software (Sartorius) was used in each case to derive kinetic parameters from the concentration series of analyte in dilution buffer that bound to the captured ligand. Association and dissociation curves were fitted to a 1:1 binding model using the data analysis software according to the manufacturer's guidelines.

[0294] The affinity and kinetic parameters (background subtracted) are shown in Figures 29A and 29B, where k on = association velocity, k dis = dissociation rate, and K D = ratio k dis / k on is the affinity constant determined by

[0295] Example 28: Inhibition of c-Kit phosphorylation in human CHO-KIT cells Standard MSD plates were coated with purified anti-human CD117 (c-Kit) antibody, sealed, shaken at 500 rpm for 10 minutes, and incubated overnight at 4°C. 96-well tissue culture-treated plates were seeded with 100,000 CHO cells / well overexpressing human c-Kit and incubated overnight at 37°C / 5% CO2. Bispecific antibodies were diluted and then added to the appropriate wells and incubated for 2 hours at 37°C / 5% CO2. The coating antibody was removed from the MSD plates, washed, and then blocked with TBST / 5% BSA for 1 hour at room temperature with shaking at 500 RPM. Diluted recombinant human SCF was added to all wells and then incubated for 10 minutes at 37°C / 5% CO2. After incubation, cells were lysed in ice-cold PBS / 0.1% Triton X-100 supplemented with phosphatase and protease inhibitors and shaken at 4°C for 5 minutes. The blocked MSD plate was washed with TBST. Cell lysates were applied to the MSD plate and shaken at 500 rpm for 1 hour at room temperature. SulfoTag-pY20 detection antibody was added to all wells and shaken at 500 rpm for 1 hour at room temperature. Results were read in 1x Read Buffer using an MSD Sector Plate Reader. Representative curves are shown in Figure 30, which demonstrate the ability of bispecific constructs (i.e., IgG and scFv pairings shown in Table 10) to inhibit c-KIT phosphorylation in human CHO-KIT cells.

[0296] Example 29: Inhibition of human mast cell degranulation Mature human mast cells were cytokine-starved overnight at 37°C / 5% CO2. The next day, mast cells were washed, resuspended in warm HEPES buffer, and transferred to a 96-well tissue culture plate. Bispecific antibodies were diluted and incubated with human mast cells for 1 hour at 37°C in air. Human IgE was then added to the human mast cell / antibody mixture for 30 minutes at 37°C in air. After IgE treatment, human SCF and goat anti-human IgE were diluted and added to the appropriate wells to induce crosslinking and incubated with human mast cells for 30 minutes at 37°C in air. After incubation, samples were diluted with PNAG solution and incubated for 90 minutes at 37°C in air. β-hexosaminidase was then read by adding glycine buffer and analyzed at OD 405 nm using a microtiter plate reader. Representative curves are shown in Figure 31, which demonstrate the ability of bispecific constructs (i.e., IgG and scFv pairings shown in Table 10) to inhibit human mast cell degranulation.

[0297] Example 30: Inhibition of proliferation of M-07e cells M-07e cells were incubated with the bispecific antibody dilutions at 37°C and 5% CO2 for 1 hour. After incubation, recombinant human SCF (R&D Systems) was added to all wells and incubated at 37°C and 6% CO2 for 6 days. After 6 days, CellTiter Glo (Promega) was added according to the kit's instructions, and luminescence resulting from cell proliferation was detected and quantified using a microtiter plate reader. Representative inhibition curves are shown in Figure 32, which demonstrate the ability of the bispecific constructs (i.e., the IgG and scFv pairings shown in Table 10) to inhibit M-07e cell proliferation.

[0298] Example 31: Membrane-associated SCF (SCF 222 Inhibition of KIT phosphorylation in M-07e cells stimulated with soluble SCF compared with control cells M-07e cells were incubated with dilutions of the bispecific antibody CDX-622. After incubation, soluble SCF and membrane-associated SCF (SCF222 ) was added and incubated according to the kit's instructions (CellTiter Glo; Promega). Luminescence as a result of cell proliferation was detected and quantified using a microtiter plate reader. Representative inhibition data are shown in Figure 33. As shown, CDX-622 inhibited the phosphorylation of KIT in M-07e cells stimulated with soluble SCF, but not SCF. 220 It blocks KIT stimulated in expressing cells more potently than KIT.

[0299] Example 32: Inhibition of mast cell activity A pilot study of mAb12 (SCF-12) was conducted in cynomolgus macaques. Prior to the start of dosing, baseline samples, including skin biopsies from the ear pinnae, were collected from two animals. mAb12 (SCF-12) was administered by slow intravenous push at a dose level of 75 mg / kg / dose on days 1 and 8. Additional skin biopsies were collected on days 30 and 57. Nanostring RNA analysis of the biopsies was performed using a non-human primate immunological gene panel along with selected mast cell and melanocyte genes. Figures 34A and 34B show decreased expression of several selected genes associated with mast cell function, reflecting significantly reduced mast cell activity.

[0300] Example 33: Histology of a Pilot Study During the course of the pilot study described in Example 32, punch biopsies from monkey ear pinnae were formalin-fixed and paraffin-embedded. Blocks were sectioned and stained with toluidine blue. Representative images of biopsy sections from animals treated with mAb12 are shown in Figure 35A (pre-treatment) and Figure 35B (day 30). Mast cells were counted, and the average data is shown in Figure 35A, and the aggregate data is shown in Figure 36B.

[0301] Example 34: Hematology Pilot Study Clinical pathology analyses were performed periodically throughout the pilot study described in Example 32. Mild hematological changes consisted of a transient decrease in mean corpuscular hemoglobin concentration and a transient increase in mean corpuscular volume without a change in mean corpuscular hemoglobin after the first dose of mAb12. The results are shown in Figures 37A, 37B, and 37C.

[0302] Example 35: Pharmacology of a Pilot Study Serum samples were collected from the monkeys over the course of the study and assayed for the presence of mAb12. Circulating levels of the monoclonal antibody are shown in Figure 38. Serum samples were also assayed for the presence of anti-drug antibodies (ADA). The results are shown in Figure 39 (the red line indicates the cutpoint of the assay).

[0303] Example 36: Inhibition of mast cell activity A pilot study of the bispecific antibody CDX-622 was conducted in cynomolgus macaques. 10 mg / kg of CDX-622 was administered by slow intravenous infusion to four cynomolgus macaques on day 1. Fluid samples were taken as indicated to determine circulating levels of test substance, anti-drug antibodies, and clinical pathology. Administration of CDX-622 did not result in significant decreases in hematological parameters (see Figures 40A-F).

[0304] Punch biopsies from the pinna of each ear were collected for histology on days 0 (pre-dose), 15, and 29 post-treatment. RNA from each biopsy was isolated and subjected to RNA quantification by Nanostring analysis. Normalized mast cell-specific gene counts are plotted as a function of baseline values, demonstrating mast cell depletion (Figures 41A-F). The housekeeping gene, β-tubulin, is shown as a control.

[0305] Example 37: Inhibition of TSLP-induced CD80 expression on human dendritic cells Human dendritic cells (DCs) were isolated from pre-frozen peripheral blood mononuclear cell leukopacks (BioIVT®) using the MACS Cell Separation Pan DC Enrichment Kit from Miltenyi Biotec. Cells were incubated overnight at 37°C and 6% CO2 with recombinant human TSLP (R&D Systems) in the presence of either medium, antibody (bispecific antibody (CDX-622) or 1D10), or isotype control. Cells were harvested, washed, and incubated with FITC-labeled CD80 (BD Biosciences). After washing, cell-associated fluorescence was determined by analysis using a FACSCanto II™ instrument (BD Biosciences) according to the manufacturer's instructions. The results are shown in Figure 42, which demonstrates that the bispecific construct CDX-622 inhibited TSLP-induced upregulation of CD80 cell surface expression on dendritic cells to the same extent as the parental mAb 1D10.

[0306] Example 38: Inhibition of TSLP binding to TSLP-R in vitro Inhibition of TSLP binding to TSLP-R in vitro by bispecific antibody CDX-622 was measured by ELISA using a method adapted from Example 23. The results are shown in Figure 43, which shows that the bispecific construct CDX-622 inhibited binding of TSLP to its cognate receptor complex (TSLPR / IL-7Rα) to the same extent as the parental mAb 1D10.

[0307] Example 39: Inhibition of TSLP-mediated cell proliferation in vitro Inhibition of TSLP-mediated cell proliferation in BaF3 cells in vitro by the bispecific antibody CDX-622 was measured using a method adapted from Example 25. The results are shown in Figure 44, which shows that the bispecific construct CDX-622 inhibited TSLP-mediated cell proliferation in vitro to the same extent as the parental mAb 1D10.

[0308] Example 40: Eosinophil survival Human eosinophils were isolated from buffy coats using the EasySep Direct Human Eosinophil Isolation Kit. Once isolated, eosinophils were plated overnight with IL-5 (0.1 ng / mL). After priming, eosinophils were treated with SCF (25 ng / mL), TSLP (1000 ng / mL), or a combination of both cytokines in the presence of either medium or CDX-622. Eosinophils were cultured at 37°C / 5% CO2 for 5 days, and survival was assessed using CellTiter-Glo. As shown in Figure 45, the bispecific construct CDX-622 inhibited eosinophil survival to a greater extent in cells treated with both chemokines (SCF and TSLP) compared to cells treated with a single chemokine (SCF or TLSP).

[0309] Example 41: SCF-induced cytokine release from human primary mast cells Human primary mast cells were plated (1 × 10 ) in 96-well cell culture-treated plates. 5 Mast cells were primed in the presence of 1000 ng / mL (1000 cells / well), IL-1α (10 ng / mL), IL-3 (10 ng / mL), and TNF-α (25 ng / mL). After priming, mast cells were treated with SCF (100 ng / mL), TSLP (5 ng / mL), or a combination of both cytokines for 24 hours in the presence of either medium, mAb, or CDX-622. After incubation, supernatants were collected, and production of IL-5 (R&D Systems) was determined by ELISA. As shown in Figure 46, the bispecific construct CDX-622 simultaneously neutralizes TSLP- and SCF-mediated mast cell activation.

[0310] Example 42: MCP-1 induction using a combination of SCF and TSLP / simultaneous blockade of SCF and TSLP LAD2 cells were incubated overnight with either 12.5 ng / mL SCF (Peprotech), 50 ng / mL TSLP (R&D Systems), or their combination. Supernatants were collected, and MCP-1 production was quantified by ELISA (R&D Systems). As shown in Figure 47, co-addition of SCF and TSLP increased the secretion of several proinflammatory cytokines and chemokines.

[0311] CDX-622, parental monoclonal antibody, and isotype control were preincubated with either 12.5 ng / mL recombinant human SCF (Peprotech), 50 ng / mL recombinant human TSLP (R&D Systems), or their combination for 20 minutes at room temperature. The mixture was then added to LAD2 cells and incubated overnight. Supernatants were collected, and MCP-1 production was quantified by ELISA (R&D Systems). Representative data (Figure 48) show that addition of anti-SCF mAb or anti-TSLP mAb inhibited cytokine release induced by their respective targets, while CDX-622 reduced their secretion to a greater extent.

[0312] Example 43: Inhibition of TSLP-induced CD80 expression on human dendritic cells Human dendritic cells (DCs) were isolated from pre-frozen peripheral blood mononuclear cell leukoplakates (BioIVT) using the MACS Cell Separation Pan DC Enrichment Kit from Miltenyi Biotec. Cells were incubated overnight with recombinant human TSLP (R&D Systems) at 37°C and 6% CO2 in the presence of either medium, antibody, or isotype control. Cells were harvested, washed, and incubated with FITC-labeled CD80 (BD Biosciences). After washing, cell-associated fluorescence was determined by analysis using a FACSCanto II™ instrument (BD Biosciences) according to the manufacturer's instructions. A representative curve is shown in Figure 49, demonstrating that CDX-622 potently inhibited TSLP-induced upregulation of CD80 cell surface expression on dendritic cells.

[0313] Table 11: Sequence Listing Overview TIFF2026508389000022.tif171159TIFF2026508389000023.tif221159TIFF2026508389000024.tif221159TIFF2026508389000025.tif221159TIFF2026508389000026.tif221159TIFF2026508389000027.tif216159TIFF2026508389000028.tif220159TIFF2026508389000029.tif219159TIFF2026508389000030.tif224159TIFF2026508389000031.tif220159TIFF2026508389000032.tif220159TIFF2026508389000033.tif221159TIFF2026508389000034.tif216159TIFF2026508389000035.tif221159TIFF2026508389000036.tif220159TIFF2026508389000037.tif220159TIFF2026508389000038.tif216159TIFF2026508389000039.tif221159TIFF2026508389000040.tif220159TIFF2026508389000041.tif220159TIFF2026508389000042.tif217159TIFF2026508389000043.tif221159TIFF2026508389000044.tif220159TIFF2026508389000045.tif220159TIFF2026508389000046.tif216159TIFF2026508389000047.tif221159TIFF2026508389000048.tif220159TIFF2026508389000049.tif220159TIFF2026508389000050.tif216159TIFF2026508389000051.tif220159TIFF2026508389000052.tif220159TIFF2026508389000053.tif220159TIFF2026508389000054.tif220159TIFF2026508389000055.tif220159TIFF2026508389000056.tif220159TIFF2026508389000057.ti f220159TIFF2026508389000058.tif216159TIFF2026508389000059.tif221159TIFF2026508389000060.tif2 20159TIFF2026508389000061.tif220159TIFF2026508389000062.tif220159TIFF2026508389000063.tif220 159TIFF2026508389000064.tif222159TIFF2026508389000065.tif222159TIFF2026508389000066.tif50159.

[0314] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A bispecific construct comprising an anti-TSLP antibody, or a binding domain thereof, linked to an anti-SCF antibody, or a binding domain thereof, wherein: (a) the anti-TSLP antibody or binding domain thereof comprises heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:6, 7, and 8, (ii) SEQ ID NOs:2, 3, and 4, (iii) SEQ ID NOs:10, 11, and 12, (iv) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and (v) light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (v) SEQ ID NOs:18, 19, and 20, (vi) SEQ ID NOs:22, 23, and 24, (vii) SEQ ID NOs:26, 27, and 28, and (viii) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof; and (b) the anti-SCF antibody or binding domain thereof comprises heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, (ii) SEQ ID NOs: 38, 39, and 40, (iii) SEQ ID NOs: 42, 43, and 44, (iv) SEQ ID NOs: 34, 35, and 36, respectively, or conservative sequence modifications thereof, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, (vi) SEQ ID NOs: 54, 55, and 56, (vii) SEQ ID NOs: 50, 51, and 52, and (viii) SEQ ID NOs: 62, 63, and 64, respectively, or conservative sequence modifications thereof; The bispecific construct.

2. A bispecific construct comprising an anti-TSLP antibody, or a binding domain thereof, linked to an anti-SCF antibody, or a binding domain thereof, wherein: (a) the anti-TSLP antibody, or binding domain thereof, comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:5, 1, 9, 13, or a sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:17, 21, 25, 29, or a sequence at least 90% identical thereto; and (b) the anti-SCF antibody or binding domain thereof comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 45, 33, 37, 41, or a sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 57, 49, 53, 61, or a sequence at least 90% identical thereto; The bispecific construct.

3. The anti-TSLP antibody or binding domain thereof is selected from the group consisting of (a) SEQ ID NOs:5 and 17, (b) SEQ ID NOs:1 and 21, (c) SEQ ID NOs:1 and 25, (d) SEQ ID NOs:1 and 29, (e) SEQ ID NOs:1 and 17, (f) SEQ ID NOs:5 and 21, (g) SEQ ID NOs:5 and 25, (h) SEQ ID NOs:5 and 29, (i) SEQ ID NOs:9 and 17, (j) SEQ ID NOs:9 and 21, (k) SEQ ID NOs:9 and 25, (l) SEQ ID NOs:9 and 29, (m) SEQ ID NOs:13 and 17, (n) SEQ ID NOs:13 and 21, (o) SEQ ID NOs:13 and 25, or (p) SEQ ID NOs:

3. The bispecific construct of claim 1 or 2, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 13 and 29, respectively.

4. 3. The bispecific construct of claim 1 or 2, wherein the anti-TSLP antibody or binding domain thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in (a) SEQ ID NOs:5 and 17, (b) SEQ ID NOs:1 and 21, (c) SEQ ID NOs:1 and 25, (d) SEQ ID NOs:1 and 17, (e) SEQ ID NOs:5 and 21, (f) SEQ ID NOs:5 and 25, (g) SEQ ID NOs:9 and 17, (h) SEQ ID NOs:9 and 21, or (i) SEQ ID NOs:9 and 25, respectively.

5. The anti-SCF antibody or binding domain thereof is selected from the group consisting of (a) SEQ ID NOs:45 and 57, (b) SEQ ID NOs:33 and 53, (c) SEQ ID NOs:33 and 57, (d) SEQ ID NOs:33 and 61, (e) SEQ ID NOs:37 and 49, (f) SEQ ID NOs:37 and 53, (g) SEQ ID NOs:37 and 57, (h) SEQ ID NOs:33 and 61, (i) SEQ ID NOs:41 and 49, (j) SEQ ID NOs:41 and 53, (k) SEQ ID NOs:41 and 57, (l) SEQ ID NOs:41 and 61, (m) SEQ ID NOs:45 and 49, (n) SEQ ID NOs:45 and 53, (o) SEQ ID NOs:33 and 49, or (p) SEQ ID NOs:

5. The bispecific construct of any one of claims 1 to 4, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 45 and 61, respectively.

6. 5. The bispecific construct of any one of claims 1 to 4, wherein the anti-SCF antibody or binding domain thereof comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in (a) SEQ ID NOs: 45 and 57, (b) SEQ ID NOs: 33 and 61, (c) SEQ ID NOs: 37 and 57, (d) SEQ ID NOs: 37 and 61, (e) SEQ ID NOs: 41 and 57, (f) SEQ ID NOs: 41 and 61, (g) SEQ ID NOs: 33 and 57, or (h) SEQ ID NOs: 45 and 61, respectively.

7. 7. The bispecific construct of any one of claims 1 to 6, wherein (a) the anti-TSLP antibody or binding domain thereof further comprises a human IgG1 constant domain, or (b) the anti-SCF antibody or binding domain thereof further comprises a human IgG1 constant domain.

8. 8. The bispecific construct of any one of claims 1 to 7, wherein (a) the anti-SCF antibody or binding domain thereof is linked to the C-terminus of the heavy chain of the anti-TSLP antibody or binding domain thereof, or (b) the anti-TSLP antibody or binding domain thereof is linked to the C-terminus of the heavy chain of the anti-SCF antibody or binding domain thereof.

9. 9. The bispecific construct of any one of claims 1 to 8, wherein (a) the anti-SCF binding domain is an scFv; or (b) the anti-TSLP binding domain is an scFv.

10. 10. The bispecific construct of any one of claims 1 to 9, wherein the anti-TSLP antibody or a binding domain thereof and the anti-SCF antibody or a binding domain thereof are genetically fused.

11. 10. The bispecific construct of any one of claims 1 to 9, wherein the anti-TSLP antibody or binding domain thereof and the anti-SCF antibody or binding domain thereof are chemically conjugated.

12. A bispecific construct comprising an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:34, 35, and 36, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:62, 63, and 64, respectively; and (b) the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and a human IgG1 constant domain; The bispecific construct.

13. (a) the anti-SCF scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:33 and 61, respectively; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 17, respectively, and a human IgG1 constant domain; 13. The bispecific construct of claim 12.

14. A bispecific construct comprising an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; and (b) the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and a human IgG1 constant domain; The bispecific construct.

15. (a) the anti-SCF scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:37 and 61, respectively; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 17, respectively, and a human IgG1 constant domain; 15. The bispecific construct of claim 14.

16. A bispecific construct comprising an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:58, 59, and 60, respectively; and (b) the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and a human IgG1 constant domain; The bispecific construct.

17. (a) the anti-SCF scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:45 and 57, respectively; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 17, respectively, and a human IgG1 constant domain; 17. The bispecific construct of claim 16.

18. A bispecific construct comprising an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:34, 35, and 36, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:62, 63, and 64, respectively; and (b) the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and a human IgG1 constant domain; The bispecific construct.

19. (a) the anti-SCF scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:33 and 61, respectively; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5 and 17, respectively, and a human IgG1 constant domain; 19. The bispecific construct of claim 18.

20. A bispecific construct comprising an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively; and (b) the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and a human IgG1 constant domain; The bispecific construct.

21. (a) the anti-SCF scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:37 and 61, respectively; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5 and 17, respectively, and a human IgG1 constant domain; 21. The bispecific construct of claim 20.

22. A bispecific construct comprising an anti-TSLP antibody linked to an anti-SCF scFv, wherein: (a) the anti-SCF scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs:58, 59, and 60, respectively; and (b) the anti-TSLP antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and a human IgG1 constant domain; The bispecific construct.

23. (a) the anti-SCF scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs:45 and 57, respectively; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5 and 17, respectively, and a human IgG1 constant domain; 23. The bispecific construct of claim 22.

24. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively; The bispecific construct.

25. (a) the anti-SCF antibody comprises heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:33 and 61, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 17, respectively; 25. The bispecific construct of claim 24.

26. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively; The bispecific construct.

27. (a) the anti-SCF antibody comprises heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:37 and 61, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 17, respectively; 27. The bispecific construct of claim 26.

28. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively; The bispecific construct.

29. (a) the anti-SCF antibody comprises heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:45 and 57, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP antibody comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 1 and 17, respectively; 29. The bispecific construct of claim 28.

30. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively; The bispecific construct.

31. (a) the anti-SCF antibody comprises heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:33 and 61, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5 and 17, respectively; 31. The bispecific construct of claim 30.

32. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 38, 39, and 40, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 62, 63, and 64, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively; The bispecific construct.

33. (a) the anti-SCF antibody comprises heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:37 and 61, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5 and 17, respectively; 33. The bispecific construct of claim 32.

34. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein: (a) the anti-SCF antibody comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises heavy chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and light chain variable region CDR1, CDR2, and CDR3 domains comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively; The bispecific construct.

35. (a) the anti-SCF antibody comprises heavy and light chain variable regions comprising the amino acid sequences set forth in SEQ ID NOs:45 and 57, respectively, and a human IgG1 constant domain; and (b) the anti-TSLP scFv comprises a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 5 and 17, respectively; 35. The bispecific construct of claim 34.

36. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein the bispecific construct comprises the amino acid sequence set forth in SEQ ID NO:65 or is encoded by the nucleotide sequence set forth in SEQ ID NO:

66.

37. A bispecific construct comprising an anti-SCF antibody linked to an anti-TSLP scFv, wherein the bispecific construct comprises the amino acid sequence set forth in SEQ ID NO:67 or is encoded by the nucleotide sequence set forth in SEQ ID NO:

68.

38. 24. The bispecific construct of any one of claims 12 to 23, wherein the anti-SCF scFv is linked to the C-terminus of the heavy chain of the anti-TSLP antibody.

39. 36. The bispecific construct of any one of claims 24 to 35, wherein the anti-TSLP scFv is linked to the C-terminus of the heavy chain of the anti-SCF antibody.

40. 36. The bispecific construct of any one of claims 12 to 35, wherein the antibody and scFv are genetically fused.

41. 36. The bispecific construct of any one of claims 12 to 35, wherein the antibody and scFv are chemically conjugated.

42. An anti-TSLP antibody or antigen-binding fragment thereof comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:2, 3, and 4, (ii) SEQ ID NOs:6, 7, and 8, (iii) SEQ ID NOs:10, 11, and 12, (iv) SEQ ID NOs:14, 15, and 16, respectively, or conservative sequence modifications thereof, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (v) SEQ ID NOs:18, 19, and 20, (vi) SEQ ID NOs:22, 23, and 24, (vii) SEQ ID NOs:26, 27, and 28, or (viii) SEQ ID NOs:30, 31, and 32, respectively, or conservative sequence modifications thereof.

43. 43. The anti-TSLP antibody or antigen-binding fragment thereof of claim 42, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, or 13, or a sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 17, 21, 25, or 29, or a sequence at least 90% identical thereto.

44. (a) SEQ ID NOs: 1 and 17, (b) SEQ ID NOs: 1 and 21, (c) SEQ ID NOs: 1 and 25, (d) SEQ ID NOs: 1 and 29, (e) SEQ ID NOs: 5 and 17, (f) SEQ ID NOs: 5 and 21, (g) SEQ ID NOs: 5 and 25, (h) SEQ ID NOs: 5 and 29, (i) SEQ ID NOs: 9 and 17, (j) SEQ ID NOs: 9 and 21, (k) SEQ ID NOs: 9 and 25, (l) SEQ ID NOs: 9 and 29, (m) SEQ ID NOs: 13 and 17, (n) SEQ ID NOs: 13 and 21, (o) SEQ ID NOs: 13 and 25, or (p) SEQ ID 44. The anti-TSLP antibody or antigen-binding fragment thereof of claim 42 or 43, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 13 and 29, respectively.

45. 44. The anti-TSLP antibody or antigen-binding fragment thereof of claim 42 or 43, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in (a) SEQ ID NOs:1 and 17, (b) SEQ ID NOs:1 and 21, (c) SEQ ID NOs:1 and 25, (d) SEQ ID NOs:5 and 17, (e) SEQ ID NOs:5 and 21, (f) SEQ ID NOs:5 and 25, (g) SEQ ID NOs:9 and 17, (h) SEQ ID NOs:9 and 21, or (i) SEQ ID NOs:9 and 25, respectively.

46. 46. ​​A bispecific construct comprising the anti-TSLP antibody, or antigen-binding fragment thereof, of any one of claims 42 to 45 linked to a second antibody, or antigen-binding fragment thereof.

47. 47. The bispecific construct of claim 46, wherein the second antibody or antigen-binding fragment thereof binds to (a) a member of the TNF superfamily (e.g., TNFα), (b) a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4), (c) an interleukin (e.g., IL-23, IL-17A, or IL-13), (d) an immunoglobulin (e.g., IgE), or (e) an integrin (e.g., integrin α4β7).

48. 48. The bispecific construct of claim 47, further comprising one or more additional binding agents.

49. An anti-SCF antibody or antigen-binding fragment thereof comprising heavy chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (i) SEQ ID NOs:34, 35, and 36, (ii) SEQ ID NOs:38, 39, and 40, (iii) SEQ ID NOs:42, 43, and 44, (iv) SEQ ID NOs:46, 47, and 48, respectively, or conservative sequence modifications thereof, and light chain variable region CDR1, CDR2, and CDR3 domains having the amino acid sequences set forth in (v) SEQ ID NOs:50, 51, and 52, (vi) SEQ ID NOs:54, 55, and 56, (vii) SEQ ID NOs:58, 59, and 60, or (viii) SEQ ID NOs:62, 63, and 64, respectively, or conservative sequence modifications thereof.

50. 50. The anti-SCF antibody or antigen-binding fragment thereof of claim 49, comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 33, 37, 41, or 45, or a sequence at least 90% identical thereto, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 49, 53, 57, or 61, or a sequence at least 90% identical thereto.

51. (a) SEQ ID NOs: 33 and 49, (b) SEQ ID NOs: 33 and 53, (c) SEQ ID NOs: 33 and 57, (d) SEQ ID NOs: 33 and 61, (e) SEQ ID NOs: 37 and 49, (f) SEQ ID NOs: 37 and 53, (g) SEQ ID NOs: 37 and 57, (h) SEQ ID NOs: 33 and 61, (i) SEQ ID NOs: 41 and 49, (j) SEQ ID NOs: 41 and 53, (k) SEQ ID NOs: 41 and 57, (l) SEQ ID NOs: 41 and 61, (m) SEQ ID NOs: 45 and 49, (n) SEQ ID NOs: 45 and 53, (o) SEQ ID NOs: 45 and 57, or (p) SEQ ID 51. The anti-SCF antibody or antigen-binding fragment thereof of claim 49 or 50, comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences set forth in SEQ ID NOs: 45 and 61, respectively.

52. 51. The anti-SCF antibody or antigen-binding fragment thereof of claim 49 or 50, comprising a heavy chain variable region and a light chain variable region, respectively, comprising the amino acid sequences set forth in (a) SEQ ID NOs:33 and 57, (b) SEQ ID NOs:33 and 61, (c) SEQ ID NOs:37 and 57, (d) SEQ ID NOs:37 and 61, (e) SEQ ID NOs:41 and 57, (f) SEQ ID NOs:41 and 61, (g) SEQ ID NOs:45 and 57, or (h) SEQ ID NOs:45 and 61.

53. A bispecific construct comprising the anti-SCF antibody or antigen-binding fragment thereof of any one of claims 49 to 52 linked to a second antibody or antigen-binding fragment thereof.

54. 54. The bispecific construct of claim 53, wherein the second antibody or antigen-binding fragment thereof binds to (a) a member of the TNF superfamily (e.g., TNFα), (b) a tumor necrosis factor (TNF) receptor (e.g., TNFRSF4), (c) an interleukin (e.g., IL-23, IL-17A, or IL-13), (d) an immunoglobulin (e.g., IgE), or (e) an integrin (e.g., integrin α4β7).

55. 55. The bispecific construct of claim 54, further comprising one or more additional binding agents.

56. 52. A composition comprising the bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or the antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, and a pharmaceutically acceptable carrier.

57. 52. A kit comprising a bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, and instructions for use.

58. 1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody variable region, wherein the antibody variable region comprises an amino acid sequence set forth in SEQ ID NO:1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, or 61.

59. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region and a light chain variable region of an antibody, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of (a) SEQ ID NOs:1 and 17, (b) SEQ ID NOs:1 and 21, (c) SEQ ID NOs:1 and 25, (d) SEQ ID NOs:1 and 29, (e) SEQ ID NOs:5 and 17, (f) SEQ ID NOs:5 and 21, (g) SEQ ID NOs:5 and 25, (h) SEQ ID NOs:5 and 29, (i) SEQ ID NOs:9 and 17, (j) SEQ ID NOs:9 and 21, (k) SEQ ID NOs:9 and 25, (l) SEQ ID NOs:9 and 29, (m) SEQ ID NOs:13 and 17, (n) SEQ ID NOs:13 and 21, (o) SEQ ID NOs:13 and 25, (p) SEQ ID NOs: NOs:13 and 29, (q) SEQ ID NOs:33 and 49, (r) SEQ ID NOs:33 and 53, (s) SEQ ID NOs:33 and 57, (t) SEQ ID NOs:33 and 61, (u) SEQ ID NOs:37 and 49, (v) SEQ ID NOs:37 and 53, (w) SEQ ID NOs:37 and 57, (x) SEQ ID NOs:33 and 61, (y) SEQ ID NOs:41 and 49, (z) SEQ ID NOs:41 and 53, (aa) SEQ ID NOs:41 and 57, (bb) SEQ ID NOs:41 and 61, (cc) SEQ ID NOs:45 and 49, (dd) SEQ ID NOs:45 and 53, (ee) SEQ ID NOs:45 and 57, or (ff) SEQ ID The isolated nucleic acid molecules comprising the amino acid sequences set forth in SEQ ID NOs:45 and 61, respectively.

60. 10. A nucleic acid molecule encoding the bispecific construct of any one of claims 1 to 41, 46 to 48, and 55 to 55, such as an isolated nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 65-69, 269-272, 274-277, 281-284, 286-289, or 350.

61. 61. The nucleic acid molecule of any one of claims 58 to 60 in the form of an expression vector.

62. 56. A method for blocking the binding of TSLP to TLSPR expressed on immune cells in a subject, comprising administering to the subject a bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, or a composition of claim 56, in an amount effective to block the binding of TSLP to TSLPR.

63. 56. A method of blocking SCF binding to c-Kit expressed on immune cells in a subject, comprising administering to the subject a bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, or a composition of claim 56, in an amount effective to block SCF binding to c-Kit.

64. 56. A method for inhibiting immune cell activation in a subject, comprising administering to the subject a bispecific construct of any of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any of claims 42 to 45 and 47 to 50, or a composition of claim 56, in an amount effective to inhibit immune cell activation in the subject.

65. 56. A method for reducing the accumulation of immune cells in an organ or tissue in a subject, comprising the step of administering to the subject a bispecific construct of any of claims 1 to 41, 46 to 48, 53 to 55, or the antibody or antigen-binding fragment thereof of any of claims 42 to 45 and 47 to 50, or the composition of claim 56, in an amount effective to reduce the accumulation of immune cells in an organ or tissue in the subject.

66. 56. A method for reducing inflammation in a subject, comprising the step of administering to the subject a bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, or a composition of claim 56, in an amount effective to reduce inflammation in the subject.

67. 56. A method for treating an inflammatory disease in a subject, comprising administering to the subject a bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, or a composition of claim 56, in an amount effective to treat the subject.

68. The following biological activities: (a) inhibition of TSLP-induced activation and / or proliferation of mast cells, dendritic cells (DCs), and / or natural killer T cells (NKTs); (b) inhibition of TSLP-induced osteoprotegerin (OPG) secretion; (c) inhibition of TSLP-induced secretion of Th2 cytokines (such as TARC, CCL22, IL-4, IL-13, or IL-5); or (d) inhibition of SCF-induced secretion of mast cells, eosinophils, type 2 innate lymphoid cells (ILC2s), and / or type 3 innate lymphoid cells (ILC3s).

68. The method of any one of claims 63 to 67, wherein the method results in at least one of the following in the subject:

69. The subject is diagnosed with a disorder of the immune system, allergic inflammation, allergic airway inflammation, DC-mediated inflammatory Th2 response, atopic dermatitis, atopic eczema, asthma, obstructive airway disease, chronic obstructive pulmonary disease, food allergy, inflammatory arthritis, rheumatoid arthritis, psoriasis, IgE-mediated disorder, rhinoconjunctivitis, fibrotic disease, disease associated with tissue remodeling, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity 68. The method of any one of claims 64-67, wherein the patient has been diagnosed with pneumonia, asthma, sclerodoma, inflammation, cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, liver fibrosis, fibromyalgia, gingival fibrosis, radiation-induced fibrosis, cardiovascular disease, gastrointestinal disease, pulmonary disease, metabolic disease (such as type 2 diabetes), neurodegenerative disease (such as Parkinson's disease), or colon cancer.

70. 70. The method of any one of claims 63-69, further comprising administration of one or more additional therapeutic agents.

71. The additional therapeutic agent may be an immunosuppressant (e.g., a corticosteroid, a nonsteroidal glucocorticoid receptor agonist, a leukotriene D4 antagonist, a leukotriene B4 antagonist, an A2A agonist, an A2B antagonist, a dopamine receptor agonist, pirfenidone, nintedanib, or an avB6 antagonist), a bronchodilator (e.g., a beta-2 adrenergic receptor agonist, a muscarinic antagonist, a short-acting beta-2 receptor agonist, a long-acting beta-2 receptor agonist, a short-acting anticholinergic, a methylxanthine, a long-acting anticholinergic), another cytokine or cytokine receptor antagonist. The method of claim 70, wherein the therapeutic agent is selected from, but is not limited to, an antagonist or antibody of a receptor (e.g., an IL-13 antagonist, an IL-6 antagonist, an IL-1, IL-33, IL-25, or TNF-α antagonist, an anti-IgE antibody, an anti-IL31 antibody, an anti-IL31R antibody, an anti-IL13 antibody, an anti-endoglin antibody, an anti-IL1b antibody, another anti-TSLP antibody, or an anti-hTSLPR antibody), an antibiotic, radiation therapy, a leukotriene antagonist (e.g., montelukast, zafirlukast, or pranlukast), a PDE4 inhibitor (e.g., roflumilast, xanthene), an antihistamine, or an antitussive.

72. 56. An in vitro method for blocking the binding of TSLP to TSLPR, comprising contacting a cell expressing TSLPR with a bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or an antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, or a composition of claim 56, in an amount effective to block the binding of TSLP to TSLPR.

73. 56. An in vitro method for blocking SCF binding to c-Kit, comprising the step of contacting a cell expressing c-Kit with the bispecific construct of any one of claims 1 to 41, 46 to 48, 53 to 55, or the antibody or antigen-binding fragment thereof of any one of claims 42 to 45 and 47 to 50, or the composition of claim 56, in an amount effective to block SCF binding to c-Kit.

74. Soluble human SCF (hSCF) 165 ), whereas membrane-bound human SCF (hSCF 222 ) with higher affinity than to IgG1.

75. A human, humanized, or chimeric antibody, or a bispecific construct thereof, that binds to an epitope comprising residue K100 of human SCF.

76. A bispecific construct comprising an anti-TSLP antibody or binding domain thereof linked to an anti-SCF antibody or binding domain thereof.

77. 77. A method for reducing mast cell activity by administering to a patient in need thereof the antibody or bispecific construct of any one of claims 74 to 76.

78. 77. A method for treating an inflammatory disorder by administering to a patient in need thereof the antibody or bispecific construct of any one of claims 74 to 76.

79. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to TSLP.

80. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a member of the TNF superfamily.

81. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a tumor necrosis factor (TNF) receptor.

82. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IL-12 and / or IL-23.

83. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-23A.

84. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-17A.

85. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-13.

86. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds IgE.

87. A construct comprising an anti-SCF antibody (or binding domain) linked to a second antibody (or binding domain) that binds to an integrin.

88. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to a member of the TNF superfamily.

89. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-12 and / or IL-23.

90. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-23A.

91. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-17A.

92. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IL-13.

93. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to IgE.

94. A construct comprising an anti-TSLP antibody (or binding domain) linked to a second antibody (or binding domain) that binds to an integrin.

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